Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones

ABSTRACT

The present invention provides dosing regimens for administering pharmacological chaperones to a subject in need thereof. The dosing regimens can be used to treat disorders caused by improper protein misfolding, such as lysosomal storage disorders.

This application is a divisional application of U.S. application Ser.No. 15/213,920, filed Jul. 19, 2016, which is a continuation-in-partapplication of U.S. application Ser. No. 14/713,821, filed May 15, 2015,now abandoned, which is a divisional application of U.S. applicationSer. No. 12/597,238, now U.S. Pat. No. 9,056,101, which is a NationalStage Application of PCT/US2008/061764, filed Apr. 28, 2008, whichclaims the benefit of U.S. Provisional Application No. 60/914,288, filedApr. 26, 2007, U.S. Provisional Application No. 61/014,744, filed Dec.18, 2007 and U.S. Provisional Application No. 61/028,105, filed Feb. 12,2008. The contents of each of these applications are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention provides a dosing regimen and rationale thereforefor the use of small molecule competitive inhibitors as pharmacologicalchaperones for the treatment of lysosomal storage diseases.

BACKGROUND

In the human body, proteins are involved in almost every aspect ofcellular function. Proteins are linear strings of amino acids that foldand twist into specific three-dimensional shapes in order to functionproperly. Certain human diseases result from mutations that causechanges in the amino acid sequence of a protein which reduce itsstability and may prevent it from folding properly. The majority ofgenetic mutations that lead to the production of less stable ormisfolded proteins are called missense mutations. These mutations resultin the substitution of a single amino acid for another in the protein.Because of this error, missense mutations often result in proteins thathave a reduced level of biological activity. In addition to missensemutations, there are also other types of mutations that can result inproteins with reduced biological activity.

Proteins generally fold in a specific region of the cell known as theendoplasmic reticulum, or ER. The cell has quality control mechanismsthat ensure that proteins are folded into their correctthree-dimensional shape before they can move from the ER to theappropriate destination in the cell, a process generally referred to asprotein trafficking. Misfolded proteins are often eliminated by thequality control mechanisms after initially being retained in the ER. Incertain instances, misfolded proteins can accumulate in the ER beforebeing eliminated.

The retention of misfolded proteins in the ER interrupts their propertrafficking, and the resulting reduced biological activity can lead toimpaired cellular function and ultimately to disease. In addition, theaccumulation of misfolded proteins in the ER may lead to various typesof stress on cells, which may also contribute to cellular dysfunctionand disease.

Lysosomal storage diseases (LSDs) are characterized by deficiencies oflysosomal enzymes due to mutations in the genes encoding the lysosomalenzymes. This results in the pathologic accumulation of substrates ofthose enzymes, which include lipids, carbohydrates, and polysaccharides.There are about fifty known LSDs to date, which include Gaucher disease,Fabry disease, Pompe disease, Tay Sachs disease and themucopolysaccharidoses (MPS). Most LSDs are inherited as an autosomalrecessive trait, although males with Fabry disease and MPS II arehemizygotes because the disease genes are encoded on the X chromosome.For most LSDs, there is no available treatment beyond symptomaticmanagement. For several LSDs, including Gaucher, Fabry, Pompe, and MPS Iand VI, enzyme replacement therapy (ERT) using recombinant enzymes isavailable. For Gaucher disease, substrate reduction therapy (SRT) alsois available in limited situations. SRT employs a small moleculeinhibitor of an enzyme required for the synthesis of glucosylceramide(the GD substrate). The goal of SRT is to reduce production of thesubstrate and reduce pathologic accumulation.

Although there are many different mutant genotypes associated with eachLSD, some of the mutations, including some of the most prevalentmutations, are missense mutations which can lead to the production of aless stable enzyme. These less stable enzymes are sometimes prematurelydegraded by the ER-associated degradation pathway. This results in theenzyme deficiency in the lysosome, and the pathologic accumulation ofsubstrate. Such mutant enzymes are sometimes referred to in thepertinent art as “folding mutants” or “conformational mutants.”

It has previously been shown that the binding of small moleculeinhibitors of enzymes associated with LSDs can increase the stability ofboth mutant enzyme and the corresponding wild-type enzyme (see U.S. Pat.Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829, and7,141,582 all incorporated herein by reference). In particular, it wasdiscovered that administration of small molecule derivatives of glucoseand galactose, which are specific, selective competitive inhibitors forseveral target lysosomal enzymes, effectively increased the stability ofthe enzymes in cells in vitro and, thus, increased trafficking of theenzymes to the lysosome. Thus, by increasing the amount of enzyme in thelysosome, hydrolysis of the enzyme substrates is expected to increase.The original theory behind this strategy was as follows: since themutant enzyme protein is unstable in the ER (Ishii et al., Biochem.Biophys. Res. Comm. 1996; 220: 812-815), the enzyme protein is retardedin the normal transport pathway (ER→Golgi apparatus→endosomes→lysosome)and prematurely degraded. Therefore, a compound which binds to andincreases the stability of a mutant enzyme, may serve as a “chaperone”for the enzyme and increase the amount that can exit the ER and move tothe lysosomes. In addition, because the folding and trafficking of somewild-type proteins is incomplete, with up to 70% of some wild-typeproteins being degraded in some instances prior to reaching their finalcellular location, the chaperones can be used to stabilize wild-typeenzymes and increase the amount of enzyme which can exit the ER and betrafficked to lysosomes.

Since some enzyme inhibitors are known to bind specifically to thecatalytic center of the enzyme (the “active site”), resulting instabilization of enzyme conformation in vitro, these inhibitors wereproposed, somewhat paradoxically, to be effective chaperones that couldhelp restore exit from the ER, trafficking to the lysosomes, hydrolyticactivity. These specific pharmacological chaperones were designated“active site-specific chaperones (ASSCs)” or “specific pharmacologicalchaperones” since they bound in the active site of the enzyme in aspecific fashion. Pharmacological chaperone therapy has potentialadvantages over ERT since a small molecule can be orally administeredand may have superior biodistribution compared to protein-basedtherapies.

Currently, three pharmacological chaperones are in human clinical trialsfor Fabry disease, Gaucher disease, and Pompe disease. Since thechaperones are competitive inhibitors of the enzymes which are deficientin these diseases, appropriate dosing regimens must be designed whichwill result in a net increase of cellular enzyme activity and notsustained inhibition of the already-deficient enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 depicts plasma PK results following administration ofisofagomine tartrate to healthy human volunteers.

FIG. 2. FIG. 2 depicts pharmacodynamic results following administrationof isofagomine tartrate to healthy human volunteers.

FIGS. 3A, 3B, and 3C. FIGS. 3A, 3B, and 3C shows the results of insilico modeling of drug plasma concentrations above and below the EC₅₀and IC₅₀ over 28 days following daily administration of IFG. In FIG. 3A,150 mg of IFG was modeled. In FIG. 3B, 25 mg of IFG was modeled. In FIG.3C, 150 mg of IFG every 4 days was modeled.

FIGS. 4A, 4B, and 4C. FIGS. 4A, 4B, and 4C show in vitro increases inGCase activity as measured using an artificial substrate in cell lysatesfollowing treatment with IFG at different concentrations for 5 days.FIG. 4A depicts GCase activity in fibroblasts; FIG. 4B depicts GCaseactivity in lymphoblasts; and FIG. 4C depicts GCase activity inmacrophages.

FIGS. 5A, 5B, and 5C. FIG. 5A shows the results of in silico modeling ofGCase accumulation rates following administration of 150 mg of IFG every2 or 3 days. FIG. 5B shows the estimated plasma PK results followingadministration of 150 mg of IFG daily for 7 days, and then 150 mg every3 or 4 days.

FIG. 5C shows the expected results of daily administration of 150 mg ofIFG every 3 days, followed by 4 days “drug free,” and also shows theexpected results of daily administration of 150 mg of IFG every 4 days,followed by 3 days “drug free.”

FIG. 6. FIG. 6 shows the results of in silico modeling of drug plasmaconcentrations above and below the EC₅₀ and IC₅₀, respectively,following administration of 150 mg of DGJ every other day.

FIG. 7. FIG. 7 shows results from 11 Fabry patients treated with DGJaccording to two specific dosing regimens.

FIG. 8. FIG. 8 is a table summarizing four dosing regimens described inExample 6.

FIG. 9. FIG. 9 is a graph of the α-GAL activity in white blood cells for8 male patients having particular missense mutations.

FIG. 10. FIG. 10 is a graph of the α-GAL activity in white blood cellsfor 9 male patients having particular missense mutations.

FIG. 11. FIG. 11 is a response summary for the dosing regimen describedin Example 6.

FIG. 12. FIG. 12 is graph demonstrating the increase in α-GAL activityin white blood cells for the three groups designated in Example 6.

FIG. 13. FIG. 13 is graph demonstrating the increase in α-GAL activityin kidney tissue for the three groups designated in Example 6.

FIG. 14. FIG. 14 is a table summarizing urine GL-3 change from baselineas described in Example 6.

FIGS. 15A and 15B. FIGS. 15A and 15B are tables summarizing GL-3Histology in specific cell types as described in Example 6.

FIG. 16. FIG. 16 is a table summarizing GL-3 kidney biopsy as describedin Example 6.

FIGS. 17A and 17B. FIGS. 17A and 17B is a graph demonstrating eGFRlevels at 48 weeks or more as described in Example 6.

FIG. 18. FIG. 18 is graph demonstrating ejection fraction as describedin Example 6.

FIG. 19. FIG. 19 is table summarizing self-reported Fabry symptoms.

FIG. 20. FIG. 20 is a table summarizing urine GL-3 change from Baselinefor female patients as described in Example 6.

FIG. 21. FIG. 21 is a table summarizing kidney biopsy GL-3 data inFemales.

FIG. 22. FIG. 22 is a table summarizing female self-reported Fabrysymptoms.

FIG. 23. FIG. 23 is a table demonstrating the effect of DNJ on NormalMouse GAA Activity as described in Example 7.

FIG. 24. FIG. 24 is a graph demonstrating FLA and GL-3 results in theskin, heart and kidney as described in Example 8.

FIG. 25. FIG. 25 is a picture of renal tubule sections and cardiacsections as described in Example 8.

FIG. 26. FIG. 26 is a graph of DGJ effect on GLA for various missensemutations as described in Example 8.

FIG. 27. FIG. 27 is a graph of demonstrating Gcase levels in Femur Boneand Bone Marrow as described in Example 9.

FIG. 28. FIG. 28 is a graph demonstrating IFG-tartrate distribution invarious tissue over time as described in Example 10.

FIG. 29. FIG. 29 is a graph comparing GL-3 amounts in rat skin, kidney,heart and plasma samples after 4 weeks of administering DGJ according tothe dosing regimens described in Example 11.

FIG. 30. FIG. 30 is a picture of skin, heart and kidney samples thathave undergone immunohistochemical staining after 4 weeks ofadministering DGJ according to the dosing regimens described in Example11 in order to visually access GL-3 reduction.

FIG. 31. FIG. 31 is graph of GLA and GL-3 levels in rat skin, heart andkidney samples after 4 weeks of administering DGJ according to thedosing regimens described in Example 12.

FIG. 32. FIG. 32 is a graph of GLA Activity in rat skin, heart, andkidney samples 0 to 7 days after withdrawal from DGJ as described inExample 13.

FIG. 33 FIG. 33 is a graph of GLA Activity in healthy males during 7days of twice daily administration of 50 and 150 mg of DGJ, and during a7-day washout period as described in Example 14.

FIG. 34 FIG. 34 shows the average LVMi changes from baseline to after6/12, 18/24 and 30/36 months of DGJ therapy, as described in Example 15.

FIG. 35 FIG. 35 shows (A) individual changes in GL-3 inclusion volumeper podocyte from baseline to after 6 months of DGJ treatment; (B)glomerulus from a patient with Fabry disease at baseline and (C) after 6months of treatment, as described in Example 15.

FIG. 36 FIG. 36 shows (A) individual changes in podocyte volume frombaseline to after 6 months of DGJ treatment; (B) correlation betweenpodocyte volume and podocyte inclusion volume after 6 months oftreatment; (C) volume fraction of GL-3 inclusions in podocytes (podocyteinclusion volume/podocyte volume) at baseline and after 6 months oftreatment, as described in Example 15.

FIG. 37 FIG. 37 shows (A) average foot process width in patients withFabry disease at baseline or after 6 months of DGJ treatment comparedwith 9 healthy controls; (B) correlation between change in foot processwidth and change in GL-3 inclusions volume per podocyte, as described inExample 15.

FIG. 38 FIG. 38 shows (A) individual changes in plasma lyso-Gb3 frombaseline to after 6 months of DGJ treatment; individual comparisonsbetween changes in plasma lyso-Gb3 with (B) changes in volume fractionof GL-3 inclusions in podocytes and (C) changes in GL-3 inclusionvolume, as described in Example 15.

FIG. 39 FIG. 39 shows independent comparisons of change in 24-hour urineprotein with (A) change in volume fraction of GL-3 inclusions inpodocytes and (B) GL-3 inclusion volume, as described in Example 15.

SUMMARY OF THE INVENTION

The present invention provides dosing regimens for administeringspecific pharmacological chaperones for the treatment of diseasesassociated with misfolded proteins (e.g. lysosomal storage disorders)and diseases which may be treated or ameliorated with thepharmacological chaperones described herein.

In a specific embodiment, dosing regimens are provided foradministration of isofagomine or a pharmacologically acceptable salt ofisofagomine to a patient for the treatment of Gaucher disease.

In one embodiment, from about 75 mg to about 300 mg of a pharmacologicalchaperone (e.g. isofagomine) is orally administered once daily for about4 to about 10 days, followed by orally administering a maintenance doseof about 75 to 225 mg of the pharmacological chaperone once every about3 to about 8 days.

In a further embodiment, the daily dose of pharmacological chaperoneadministered is about 125 to 225 mg/day (e.g. about 150 mg/day), and isadministered for about 5 to about 8 days (e.g. about 7 days).

In a further embodiment, the maintenance dose of the pharmacologicalchaperone (e.g. isofagomine) administered is about 125 mg to about 175mg, and is administered about every 4-7 days. In yet a furtherembodiment, the maintenance dose administered is about 150 mg, which isadministered every 4 days. In an alternate, embodiment, the maintenancedose administered is about 150 mg, which dose is administered every 7days.

In a specific embodiment, the present invention provides a method ofadministering isofagomine or a pharmacologically acceptable salt to apatient for the treatment of Gaucher disease by orally administeringabout 150 mg of isofagomine once daily for about 7 days, followed byorally administering a maintenance dose of about 150 mg of isofagomineabout once every 7 days.

The present invention also provides a method of administeringisofagomine or a pharmacologically acceptable salt to a patient in needthereof for the treatment of Gaucher disease, by orally administeringbetween about 75 mg to about 300 mg about every 2-3 days.

In one embodiment, the dose of isofagomine administered is between about125 mg to about 225 mg. In another embodiment, the dose of isofagomineadministered is about 150 mg. In a specific embodiment, about 150 mg ofisofagomine tartrate is administered about every 3 days.

In a particular embodiment of the invention, the isofagomine saltadministered is isofagomine tartrate.

In one embodiment, from about 75 to about 300 mg of a pharmacologicalchaperone is orally administered once daily for about 4 to about 10days, followed by a first washout period in which the pharmacologicalchaperone is not administered for about 1 to about 10 days, followed byorally administering a maintenance dose of about 75 to 300 mg of thepharmacological chaperone once every about 1 to about 8 days, followedby a second washout period in which the pharmacological chaperone is notadministered for about 1 to about 10 days.

In a further embodiment, the daily dose administered is about 125 to 225mg/day, and is administered for about 5 to about 8 days. In a stillfurther embodiment, the daily dose administered is about 225 mg/day, andis administered for about 7 days.

In a further embodiment, the first washout period in which thepharmacological chaperone is not administered is about from about 2 daysto about 8 days. In a specific embodiment, the first washout period inwhich the pharmacological chaperone is not administered is about 7 days.

In a further embodiment, the maintenance dose administered is from about125 mg to about 275 mg, and is administered once a day for about 4-7days. In yet a further embodiment, the maintenance dose administered isabout 225 mg, which dose is administered once a day for about 3 days.

In an alternative embodiment, no maintenance dose is administered.

In a further embodiment, the second washout period in which thepharmacological chaperone is not administered is from about 2 days toabout 8 days. In a specific embodiment, the second washout period inwhich the pharmacological chaperone is not administered is about 4 days.In an alternative embodiment, there is no second washout period in whichno pharmacological chaperone is administered.

In a further embodiment, the daily dose and the first washout periodoccurs over a period of time from about 1 week to about 30 weeks, offrom about 5 weeks to about 25 weeks.

In a further embodiment, the daily dose and the first washout periodoccurs over a period of time from about 5 weeks to about 25 weeks.

In a specific embodiment, the daily dose and the first washout periodoccurs over a period of time of about 24 weeks.

In an alternative embodiment, the daily dose and the first washoutperiod occurs over a period of time of about 2 weeks.

In a further embodiment, the maintenance dose and the second washoutperiod occurs over a period of time from about 1 week to about 30 weeks.

In a further embodiment, the daily dose and the first washout periodoccurs over a period of time from about 5 weeks to about 25 weeks.

In a specific embodiment, the maintenance dose and the second washoutperiod occurs over a period of time of about 22 weeks.

In yet a further embodiment, the patient does not ingest any food (i.e.fasts) prior to and following the administration of a pharmacologicalchaperone for a period of between about 0.5 and about 24 hours, or fromabout 1 hour to about 12 hours (e.g. about 2 hours).

In a further embodiment, the pharmacological chaperone is isofagomine ora pharmacologically acceptable salt thereof (e.g. isofagomine tartrate).

In a specific embodiment, the present invention provides a method ofadministering isofagomine or a pharmacologically acceptable salt to apatient for the treatment of Gaucher disease by orally administeringabout 225 mg of isofagomine or a pharmacologically acceptable salt oncedaily for about 7 days, followed by a first washout period in which noisofagomine or pharmacologically acceptable salt is administered forabout 7 days, followed by orally administering a maintenance dose ofabout 225 mg of isofagomine or pharmacologically acceptable salt onceeach day for about 3 days, followed by a second washout period in whichno isofagomine or pharmacologically acceptable salt is administered forabout 4 days, wherein the maintenance dose and the second washout periodrepeats for a period of 22 weeks.

In another specific embodiment, the present invention provides a methodof administering isofagomine or a pharmacologically acceptable salt to apatient for the treatment of Gaucher disease by orally administeringabout 225 mg of isofagomine or a pharmacologically acceptable salt oncedaily for about 7 days, followed by a washout period in which noisofagomine or pharmacologically acceptable salt is administered forabout 7 days, wherein the administration of the daily dose and thewashout period repeats for a period of 24 weeks.

In a particular embodiment of the invention, the isofagomine saltadministered is isofagomine tartrate.

The present invention also provides specific dosing regimens for theadministration of 1-deoxygalactonojirimycin or salts thereof for thetreatment of Fabry disease. The present invention also provides specificdosing regimens for the administration of 1-deoxygalactonojirimycin forreducing left ventricular mass index (LVMi) or podocyteglobotriaosylceramide (GL-3) in a patient having Fabry disease. Inparticular embodiments, the patient has left ventricular hypertrophy(LVH) prior to initiating administration of the1-deoxygalactonojirimycin or salt thereof.

In one embodiment of the invention, DGJ hydrochloride is orallyadministered daily from about 4 to about 10 days, or from about 5 toabout 8 days, or for about 7 days, followed by administration of amaintenance dose about every 2 days to about every 3 days.

In this embodiment, the daily dose will be in a range from about 200 mgto about 500 mg per day, or from about 250 mg to about 300 mg per day,or about 250 mg per day.

In the foregoing embodiments, the maintenance dose administered every 2to 3 days will be in a range from about 75 mg to about 225 mg, or, fromabout 100 mg to about 200 mg, or, in a specific embodiment, about 150mg.

In another embodiment, 1-deoxygalactonojirimycin is administered dailyfrom about 4 to about 14 days, or from about 5 to about 10 days, or in aparticular embodiment, for about 7 days, at a dose in a range from about200 mg to about 500 mg per day, or from about 250 mg to about 300 mg perday, or about 250 mg per day.

Following the 4-14 day period, a daily maintenance dose is administeredwhich is in a range from about 25 to 50 mg, or about 25 mg per day.

In another embodiment, interval dosing about every 2-3 days iscontemplated. In this embodiment, between about 50 mg to about 300 mg1-deoxygalactonojirimycin or salt thereof is administered at eachinterval, or from about 125 mg to about 225 mg at each interval, orabout 150 mg at each interval. In specific embodiments, DGJhydrochloride will be administered at 50 mg, 150 mg or 250 mg every 2days. In other specific embodiments, about 123 mg free base equivalent(FBE) of DGJ or a salt thereof is administered every 2 days, such asabout 123 mg of DGJ or about 150 mg of migalastat hydrochloride.

In a further embodiment, 1-deoxygalactonojirimycin will be orallyadministered 50 mg per day for two weeks, followed by 200 mg per day fortwo weeks, followed by 500 mg per day for two weeks and followed by 50mg per day for the duration of treatment.

DETAILED DESCRIPTION

The present invention provides dosing regimens for the administration ofspecific pharmacological chaperones for the treatment of a diseaseassociated or caused by one or more misfolded proteins, for example, alysosomal storage disorder. The dosing regimens described in thisapplication may also be used to treat any disease or condition which maybe treated or ameliorated with use of a pharmacological chaperone,including but not limited to, Parkinson's Disease, and Alzheimer'sDisease. For example, specific dosing regimens of isofagomine and1-deoxygalactonojirimycin are provided for the treatment of Gaucherdisease and Fabry disease, respectively, and an in silico model isprovide that can be used to predict dosing regimens for other diseasesin which the pharmacological chaperone is a viable treatment option.

Definitions

“Gaucher disease” refers to Type 1, Type 2, and Type 3 Gaucher disease.

“Fabry disease” refers to classical Fabry disease, late-onset Fabrydisease, and hemizygous females having mutations in the gene encodingα-galactosidase A.

“Pompe disease” or “glycogen storage disease type II” includesinfantile-onset, non-classical infantile-onset, and adult-onset disease.

As used herein, the term “pharmacological chaperone,” or sometimes“specific pharmacological chaperone” (“SPC”), refers to a molecule thatspecifically binds to a protein, such as an enzyme, and has one or moreof the following effects: (i) inducing a stable molecular conformationof the protein; (ii) promoting trafficking of the protein from the ER toanother cellular location, preferably a native cellular location, i.e.,preventing ER-associated degradation of the protein; (iii) preventingaggregation of unstable proteins; (iv) restoring or increasing at leastpartial wild-type function and/or activity to the protein; and/orimproving the phenotype or function of the cell harboring the protein.Thus, a pharmacological chaperone is a molecule that binds to a targetprotein, resulting in protein stabilization, trafficking,non-aggregation, and/or increasing activity of the protein. As usedherein, this term does not refer to endogenous chaperones, such as BiP,or to non-specific agents which have demonstrated non-specific chaperoneactivity, such as glycerol, DMSO or deuterated water, which aresometimes called “chemical chaperones” (see Welch et al., Cell Stressand Chaperones 1996; 1(2):109-115; Welch et al., Journal ofBioenergetics and Biomembranes 1997; 29(5):491-502; U.S. Pat. Nos.5,900,360; 6,270,954; and 6,541,195).

In various embodiments “pharmacological chaperone” or “specificpharmacological chaperone” (“SPC”) includes only active-site specificchaperones that bind to an enzyme or other protein in a competitivemanner. Unless stated otherwise, however, the “pharmacologicalchaperone” or “specific pharmacological chaperone” (“SPC”) is understoodto encompass chaperones that bind the enzyme in areas in addition to theactive site and also encompasses chaperones that bind in both acompetitive and non-competitive manner Unless specified otherwise, anyreference to administration of a pharmacological chaperone shall referto oral administration. Any reference to administration amounts ofpharmacological chaperone shall refer to oral administration amounts ofpharmacological chaperone.

As used herein, the terms “enhance protein activity” or “increaseprotein activity” refer to increasing the amount of polypeptide thatadopts a stable conformation in a cell contacted with a pharmacologicalchaperone specific for the protein, relative to the amount in a cell(preferably of the same cell-type or the same cell, e.g., at an earliertime) not contacted with the pharmacological chaperone specific for theprotein. In one embodiment, the cells do not express a mutantpolynucleotide encoding a polypeptide that is deficient with respect tothe folding and/or processing of a polypeptide in the ER. In anotherembodiment, the cells do express a mutant polynucleotide encoding apolypeptide e.g., a conformational mutant. Thus, the aforementionedterms also mean increasing the efficiency of transport of a wild-typepolypeptide to its native location in a cell contacted with apharmacological chaperone specific for the protein, relative to theefficiency of transport of a wild-type polypeptide in a cell (preferablyof the same cell, e.g., at an earlier time, or the same cell type as acontrol) not contacted with the pharmacological chaperone specific forthe protein.

The term “Vmax” refers to the maximum initial velocity of an enzymecatalyzed reaction, i.e., at saturating substrate levels. The term “Km”is the substrate concentration required to achieve ½ Vmax.

The term “AUC” represents a mathematical calculation to evaluate thebody's total exposure over time to a given drug. In a graph plotting howconcentration in the blood after dosing, the drug concentration variablelies on the y-axis and time lies on the x-axis. The area between a drugconcentration curve and the x-axis for a designated time interval is theAUC. AUCs are used as a guide for dosing schedules and to comparedifferent drugs' availability in the body.

The term “Cmax” represents the maximum plasma concentration achievedafter dosing.

The term “Tmax” represents the time to maximum plasma concentration(Cmax).

The term “Ki” refers the dissociation constant of the enzyme-inhibitorcomplex, i.e., the concentration required to inhibit half-maximal enzymeactivity. A low Ki means there is a high binding affinity of the drug tothe enzyme.

The term “EC₅₀” refers to is the concentration of a drug which induces adesired response halfway between the baseline and maximum, i.e., theconcentration at which 50% of its maximal effect is observed. Accordingto the present invention, the EC₅₀ is the concentration at which half ofthe observed maximal increase in enzyme activity occurs under a specificset of conditions.

The term “IC₅₀” represents the concentration of a drug that is requiredfor 50% enzyme inhibition in vitro in cells.

The terms “therapeutically effective dose” and “effective amount” referto the amount of the specific pharmacological chaperone that issufficient to result in a therapeutic response. A therapeutic responsemay be any response that a user (e.g., a clinician) will recognize as aneffective response to the therapy, including the foregoing symptoms andsurrogate clinical markers. Thus, a therapeutic response will generallybe an amelioration of one or more symptoms of a disease or disorder,e.g., Gaucher disease, such as those known in the art for the disease ordisorder, e.g., for Gaucher disease.

Non-limiting examples of improvements in surrogate markers for Gaucherdisease are disclosed in U.S. Ser. No. 60/911,699, hereby incorporatedby reference, and include increases in GCase levels or activity;increased trafficking of GCase from the ER to the lysosome; decreases inthe presence of lipid-laden macrophages (“Gaucher macrophages”);decreased levels of chitotriosidase; decreased levels of liver enzymes;decreased levels of pulmonary chemokine PARC/CCL18; decreased levels ofplasma α-synuclein; decreased levels of angiotensin converting enzyme(ACE) and total acid phosphatase; decreased splenomegaly andhepatomegaly, improvements in bone complications (including osteopenia,lytic lesions, pathological fractures, chronic bone pain, acute bonecrises, bone infarcts, osteonecrosis, and skeletal deformities),improvements in immunological defects such as anemia, thrombocytopenia,leukopenia, hypergammaglobulinemia, increased amount of T-lymphocytes inthe spleen, decreased B cell hyperproliferation and plasmacytosis,decreased levels of inflammatory cytokines including TNF-α, IL-1β, IL-6,IL-8, IL-17, MIP-1a and VEGF, improvements in neutrophil chemotaxis;decreased pulmonary hypertension; and decreased levels of bone-specificalkaline phosphatase, improvements in neurological symptoms such ashorizontal gaze, myoclonic movements, corneal opacity, ataxia, dementia,spasticity; seizures, auditory impairment; cognitive impairment, andneurodegeneration.

Non-limiting examples of improvements in surrogate markers for Fabrydisease include increases in α-GAL levels or activity in cells (e.g.,fibroblasts) and tissue; reductions in of GL-3 accumulation; decreasedplasma concentrations of homocysteine and vascular cell adhesionmolecule-1 (VCAM-1); decreased GL-3 accumulation within myocardial cellsand valvular fibrocytes; reduction in cardiac hypertrophy (especially ofthe left ventricle), amelioration of valvular insufficiency, andarrhythmias; amelioration of proteinuria; decreased urinaryconcentrations of lipids such as CTH, lactosylceramide, ceramide, andincreased urinary concentrations of glucosylceramide and sphingomyelin(Fuller et al., Clinical Chemistry. 2005; 51: 688-694); the absence oflaminated inclusion bodies (Zebra bodies) in glomerular epithelialcells; improvements in renal function; mitigation of hypohidrosis; theabsence of angiokeratomas; and improvements hearing abnormalities suchas high frequency sensorineural hearing loss progressive hearing loss,sudden deafness, or tinnitus. Improvements in neurological symptomsinclude prevention of transient ischemic attack (TIA) or stroke; andamelioration of neuropathic pain manifesting itself as acroparaesthesia(burning or tingling in extremities). Accordingly, the dosing regimensdescribed herein can be used to improve any of these surrogate markers,such as reducing left ventricular mass index (LVMi) or reducing GL-3accumulation in renal podocytes.

Non-limiting examples of improvements in surrogate markers for Pompedisease include increases in α-glucosidase, decreased glycogenaccumulation, decreased hypotonia, improvements in muscle function andmobility, including improved exercise tolerance, decreased macroglossia,reduction in cardiomegaly and hepatosplenomegaly, improvements inrespiratory function, improvements in swallowing, sucking or feeding,and amelioration of sleep apnea,

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a human Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin, 18th Edition, or other editions.

Isofagomine (IFG) refers to the compound(2R,3R,4R)-5-(hydroxymethyl)-piperidine-3,4-diol. Isofagomine isdescribed in U.S. Pat. Nos. 5,863,903 and 5,844,102. Isofagominetartrate has recently been described in U.S. application Ser. No.11/752,658, which is hereby incorporated by reference, and has beenassigned CAS number 919364-56-0. Isofagomine also may be prepared in theform of other acid addition salts made with a variety of organic andinorganic acids. Such salts include those formed with hydrogen chloride,hydrogen bromide, methanesulfonic acid, sulfuric acid, acetic acid,trifluoroacetic acid, oxalic acid, maleic acid, benzenesulfonic acid,toluenesulfonic acid and various others (e.g., nitrate, phosphate,borates, citrates, benzoates, ascorbates, salicylates and the like).Such salts can be formed as known to those skilled in the art.Isofagomine also may form crystals with alkali metals such as sodium,potassium and lithium, with alkaline earth metals such as calcium andmagnesium, with organic bases such as dicyclohexylamine, tributylamine,pyridine and amino acids such as arginine, lysine and the like. Suchcrystals can be formed as known to those skilled in the art.

Other potential chaperones for Gaucher disease are described in pendingU.S. patent application Ser. Nos. 10/988,428, and 10/988,427, both filedNov. 12, 2004). Such compounds include glucoimidazole((5R,6R,7S,8S)-5-hydroxymethyl-5,6,7,8-tetrahydroimidazo[1,2a]pyridine-6,7,8-triol).

“1-deoxygalactonojirimycin” (DGJ) refers to(2R,3S,4R,5S)-2-(hydroxymethyl) piperdine-3,4,5-triol, also known asmigalastat. This term includes both the free base and any salt forms.The hydrochloride salt of DGJ is known as migalastat hydrochloride. Asused herein, the term “free base equivalent” or “FBE” refers to theamount of DGJ present in the DGJ or salt thereof. In other words, theterm “FBE” means either an amount of DGJ free base, or the equivalentamount of DGJ free base that is provided by a salt of DGJ. For example,due to the weight of the chloride anion, 150 mg of DGJ HCl only providesas much DGJ as 123 mg of the free base form of DGJ. Other salts willhave different conversion factors, depending on the molecular weight ofthe counter ion.

Other chaperones for α-GAL are described in U.S. Pat. Nos. 6,274,597,6,774,135, and 6,599,919 to Fan et al., and includeα-3,4-di-epi-homonojirimycin, 4-epi-fagomine, andα-allo-homonojirimycin, N-methyl-deoxygalactonojirimycin,β-1-C-butyl-deoxygalactonojirimycin, and α-galacto-homonojirimycin,calystegine A3, calystegine B2, N-methyl-calystegine A3, andN-methyl-calystegine B2.

“1-deoxynojirimycin” (DNJ) refers to (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol. This term includes both the free base and anysalt forms, particularly the hydrochloride salt.

The term “substantially equal duration” refers to a period of time thatis at least within ±1 day of a given period of time. For example, 3 daysis a substantially equal duration when compared to 4 days, and viceversa. Thus embodiments below that call for daily dosing for three daysand a washout period of four days would be considered an example ofdaily dosing for a period of time followed by a washout period ofsubstantially equal duration.

Substantially equal duration also encompasses equal duration. Thus 7days is a substantially equal duration as 7 days. Thus embodiments belowthat call for daily dosing for seven days and a washout period of sevendays would be considered an example of daily dosing for a period of timefollowed by a washout period of substantially equal duration.

Although the dosing regimens described in this application are describedlargely in reference to lysosomal storage diseases, it is understoodthat other conditions that are caused by, or aggravated by misfoldedproteins may be treated using the dosing regimens described herein.Also, any disease or condition that may be treated or ameliorated withthe pharmacological chaperones described in this application, includingbut not limited to Alzheimer's Disease and Parkinson's Disease, may betreated using the dosing regimens of the present application.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

Gaucher Disease

Gaucher disease (GD) is a lysosomal storage disorder caused bydiminished activity of a key metabolic enzyme, β-glucocerebrosidase(GCase). The reduced activity of GCase leads to the accumulation ofglycosphingolipids called glucocerebrosides inside the lysosomes incells, in particular, macrophages of the liver, bone marrow, and spleen.Patients with GD exhibit hematological manifestations such as anemia andthrombocytopenia, as well as hepatosplenomegaly, skeletal impairment,and in some cases neurological impairment. The symptoms, severity, andage of onset depend in part on the mutations underlying the disease;over 200 mutations in the GBA gene have been identified, but fourmutations are found in the majority of patients. Two of these mutations,N370S and L444P, are amino acid substitutions that are found in morethan 90% of the Gaucher population. The other two mutations (84insG andIVS2) are DNA insertion and deletion mutations, respectively.

From a clinical perspective, GD has been classified into three subtypes:type 1 (non-neuronopathic), type 2 (infantile acute neuronopathic), andtype 3 (subacute neuronopathic). Type 1 disease is most often associatedwith the N370S mutation, but type 3 disease most often presents inpatients who carry the L444P mutation. Patients with type 1 Gaucherdisease, the most common subtype, display a wide range of symptoms.These symptoms include splenomegaly, hepatomegaly, anemia,thrombocytopenia, bone complications (including osteopenia, lyticlesions, pathological fractures, chronic bone pain, acute bone crises,bone infarcts, osteonecrosis, and skeletal deformities), and in a smallnumber of patients, interstitial lung disease and pulmonaryhypertension. Type 2 GD presents in infancy and is characterized by arapid neurodegenerative course with widespread visceral involvement.Failure to thrive and stridor because of laryngospasm are commonlyobserved, and death caused by progressive psychomotor degenerationoccurs within the first 2 to 3 years of life. Type 3 GD presents aroundpreschool age and is characterized by visceral and bone involvement, inaddition to neurological symptoms such as abnormal eye movements,ataxia, seizures, and dementia. The neurological symptoms usually appearlater in life, and patients often survive until their third or fourthdecade. The most prominent difference between the three types areneurological involvement, which is absent in type 1 and present in types2 and 3. The rate of disease progression is slow in type 1, rapid intype 2, and intermediate in type 3.5.

Current treatment options for GD include enzyme replacement therapy(ERT) and substrate reduction therapy (SRT). These therapies have beenshown to address the major hematological defects and reduce organ volumein most patients. However, neither is approved to treat the neurologicalsymptoms or skeletal symptoms of Gaucher disease.

Nonclinical toxicology studies conducted in rats and monkeys have shownthat repeated dosing with the pharmacological chaperone isofagominetartrate (IFG) is generally safe and well tolerated. IFG is animinosugar that functions as a selective pharmacological chaperone ofGCase that is less stably folded as a result of missense mutations.Current data suggest that IFG may work by stabilizing mutant forms ofGCase in the endoplasmic reticulum and promoting proper trafficking ofthe enzyme to the lysosome (Steet et al., PNAS. 2006; 103: 13813-18;Lieberman et al., Nature Chem Biol. 2007; 3(2):101-7). In the lysosome,when the pharmacological chaperone dissociates from the enzyme, theenzyme can perform its normal function, which is to catalyze thebreakdown of glucosylceramide (GlcCer), the GCase substrate. Studieshave shown that treatment with IFG increases GCase total cellular enzymelevels in vitro, increases GCase trafficking to the lysosome infibroblasts of GD patients, and increases tissue GCase activity andreduces plasma levels of chitinase and immunoglobulin G (IgG) in a mousemodel of GD. These results, along with results from early clinicalstudies in patients, strongly support the use of IFG tartrate inpatients with GD resulting from missense mutations in the GBA gene.

Fabry Disease

Fabry disease is a lysosomal storage disorder resulting from adeficiency in the lysosomal enzyme α-galactosidase A (α-GAL). Symptomscan be severe and debilitating, including kidney failure and increasedrisk of heart attack and stroke. The deficiency of α-GAL in Fabrypatients is caused by inherited genetic mutations. Certain of thesemutations cause changes in the amino acid sequence of α-GAL that mayresult in the production of α-GAL with reduced stability that does notfold into its correct three-dimensional shape. Although α-GAL producedin patient cells often retains the potential for some level ofbiological activity, the cell's quality control mechanisms recognize andretain misfolded α-GAL in the endoplasmic reticulum, or ER, until it isultimately moved to another part of the cell for degradation andelimination. Consequently, little or no α-GAL moves to the lysosome,where it normally breaks down GL-3. This leads to accumulation of GL-3in cells, which is believed to be the cause of the symptoms of Fabrydisease. In addition, accumulation of the misfolded α-GAL enzyme in theER may lead to stress on cells and inflammatory-like responses, whichmay contribute to cellular dysfunction and disease.

The clinical manifestations of Fabry disease span a broad spectrum ofseverity and roughly correlate with a patient's residual α-GAL levels.The majority of currently treated patients are referred to as classicFabry disease patients, most of whom are males. These patientsexperience disease of various organs, including the kidneys, heart andbrain, with disease symptoms first appearing in adolescence andtypically progressing in severity until death in the fourth or fifthdecade of life. A number of recent studies suggest that there are alarge number of undiagnosed males and females that have a range of Fabrydisease symptoms, such as impaired cardiac or renal function andstrokes, that usually first appear in adulthood. Individuals with thistype of Fabry disease, referred to as later-onset Fabry disease, tend tohave higher residual α-GAL levels than classic Fabry disease patients.Individuals with later-onset Fabry disease typically first experiencedisease symptoms in adulthood, and often have disease symptoms focusedon a single organ, such as enlargement of the left ventricle orprogressive kidney failure. In addition, later-onset Fabry disease mayalso present in the form of strokes of unknown cause.

Similar to IFG for GCase, DGJ has been show to bind in the active siteof α-GAL and increase its activity in vitro and in vivo (see Example 5).

Dosing Considerations for Pharmacological Chaperones

According to the present invention, dosing is determined using asimplified model which depends on certain observable factors identifiedby in vitro and in vivo evaluation. Such factors include thepharmacokinetics of the candidate pharmacological chaperone in plasmaand tissue, the rate of enzyme accumulation in the lysosome; the rate ofenzyme turnover (half life in the lysosome); and the binding affinity ofdrug to enzyme as determined in vitro. The rationale for using theforegoing parameters to model dosing regimens was determined usingisofagomine tartrate following pre-clinical studies in animals, andPhase I and Phase II trials in humans, as well as in vitro testing, asdescribed below.

TABLE 1 PK considerations PD considerations Cmax Rate of enzymeaccumulation Tmax (plasma and tissue) Rate of enzyme turnover (lysosomalhalf-life) Dosing interval Time above the EC₅₀ Drug half-life (plasmaand tissue) Time below the IC₅₀ Emax Dosing interval

Pharmacokinetics and Pharmacodynamics of IFG Tartrate.

In the Phase 1 trials to evaluate the safety of IFG, a candidatechaperone for GCase, in healthy adult subjects, single doses of up to300 mg, and repeated doses of up to 225 mg/d for 7 days wereadministered orally in randomized, double-blind, placebo controlledstudies. In the multiple-dose study, three cohorts of 8 subjects (6active and 2 placebos per cohort) received daily oral doses of 25, 75,or 225 mg IFG or placebo for 7 days, with a treatment-free safetyevaluation period of 7 days. Blood samples were collected forpharmacokinetic analysis before the initial drug administration on Day1, before the 5th, 6th and 7th doses (on Days 5, 6 and 7) (for Cmindetermination), and at the following times after the 1st (Day 1) and 7th(Day 7) doses: 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, and 24hours. In addition, a single blood sample was collected 48 hours afterthe last dose (Day 9) and assayed for the presence of IFG. In addition,blood samples were collected for pharmacodynamic measurements, i.e.,analysis of WBC GCase levels, before dosing on Day 1, Day 3, Day 5, andDay 7, and at return visits on Day 9, Day 14 and Day 21.

In the multiple-dose study, after 7 days of oral administration, thepharmacokinetic behavior was found to be linear with dose, with nounexpected accumulation of IFG. Mean plasma levels (Tmax) peaked atabout 3.4 hr. (SEM: 0.6 hr.) and the plasma elimination half-life wasabout 14 hr. (SEM: 2 hr.) (FIG. 1).

Importantly, healthy subjects receiving IFG showed a dose-dependentincrease in GCase levels in white blood cells during the 7-day treatmentperiod, in most cases peaking on day 7 of treatment, followed by a moregradual decrease in enzyme levels upon removal of the drug and a returnto near baseline levels by 14 days after the last dose (FIG. 2). Themaximum increase in enzyme level achieved was approximately 3.5-foldabove baseline levels. The lowest daily dose that achieved the maximumrate of GCase accumulation over 7 days was about 75 mg.

Based on results from the foregoing multiple-dose study and in vitrocell-based assays (healthy human skin fibroblasts), the followingobservations were made relating to the PK and PD properties of IFG.

TABLE 2 Plasma PK and PD Cmax (μM)  1.7 (150 mg IFG) 0.31 (25 mg IFG)Tmax (hrs) 3 Half-life (hrs) 14 EC₅₀ (μM) (cellular) 0.3 IC₅₀ (μM)(purified enzyme; pH 5.2) 0.03

Long-Term Maintenance of Elevated Enzyme Levels.

Since pharmacological chaperones, such as IFG, are potent inhibitors ofthe intended target enzymes, it was hypothesized that a dosing regimeninvolving “peaks” and “troughs” would be necessary to prevent sustainedinhibition of the target enzyme. Accordingly, the need for non-dailydosing as opposed to daily dosing appeared likely, in which the goalwould be to achieve plasma concentrations of the drug that are initiallyabove the cellular EC₅₀ (as determined in vitro cellular enzyme activityassays) for some period of time, so as to maximize the amount of enzymethat is trafficked to lysosomes, followed by some period of time wherethe concentration of drug falls below the IC₅₀ (as determined in vitrousing cell lysate at the lysosomal pH of 5.2). Accordingly, a simplemodel was devised wherein certain PK and PD parameters could be used toestimate dosing regimens that would both (i) achieve a plasmaconcentration above the EC₅₀ and (ii) permit the plasma concentration tofall below the IC₅₀.

In brief, the above parameters were used to estimate the plasmaconcentration over time using different dosing regimens based on theexponential terminal elimination half-life of IFG. This was followed bya determination of if and for how long the resulting plasmaconcentrations would be above the EC₅₀ or below the IC₅₀.

IC₅₀ and EC₅₀ Considerations

Based on the foregoing, it was determined that a dosing regimen in which150 mg of IFG was administered once a day would result in plasmaconcentrations that reach or exceed the observed EC₅₀ for GCase, therebypromoting chaperoning (trafficking from the ER to the lysosome) for asignificant period of time. However, at this daily dose, the plasmaconcentration is not expected to fall below the IC₅₀ for GCase (FIG.3A), which is required for maximal turnover of the accumulatedsubstrate. Accordingly, this dosing regimen may not provide the optimalresponse in vivo because they it may not permit substrate clearance.

Consideration of Dose (Cmax)

Lowering the dose to 25 mg of IFG once daily (for 28 days) is expectedto reduce the time above the EC₅₀, but concentrations below the IC₅₀ arestill not obtained (FIG. 3B). Thus, it was proposed that longerintervals at a higher dose should be used to maximize the time aboveEC₅₀ while allowing the concentration to drop below the IC₅₀ for aperiod of time.

Dosing Interval Considerations

In view of the foregoing, administration of 150 mg IFG every 4 days (for28 days) is predicted to provide plasma concentrations above and belowthe EC₅₀ and IC₅₀, respectively, for nearly equal periods of time (FIG.3C). It has been determined experimentally that maximum chaperoning(Emax) in Gaucher patient-derived fibroblasts, lymphoblast andmacrophages occurs in a range from about 10-100 μM IFG (FIG. 4). Thus,it is anticipated that the rate of GCase accumulation during the “aboveEC₅₀” time period will increase as Cmax approaches Emax.

Initial Enzyme Build-Up Phase

Accordingly, using the simplified model, it was discovered thatadministering a daily dose of IFG for an initial period of time wouldachieve the goal of maximizing the amount of GCase trafficking tolysosomes, i.e., the dose would result in plasma concentrations ofchaperone above the EC₅₀. During this period, this dose would permitspecific binding to the enzyme, increase its stability, and inducetrafficking and localization of the enzyme to the lysosomes. Thisinitial dose is referred to as the “Initial Enzyme Build-Up Phase.”

Assuming IFG has no effect on GCase's rate of synthesis, GCase's rate ofaccumulation or “build up” is determined by the difference between theamount of enzyme trafficked from the ER to the lysosome and the amountof enzyme lost due to its rate of turnover in the lysosome. Thus, GCaselevels in the lysosome will increase when the amount of GCase traffickedto the lysosome exceeds the amount of enzyme lost due to turnover, whilelysosomal levels of GCase will decrease if the amount of enzymetrafficked to the lysosome is not sufficient to replace the amount ofenzyme lost due to turnover. Since the amount of GCase trafficked to thelysosome is dependent upon the concentration of IFG (in the ER), the netchange in lysosomal GCase levels is therefore a function of IFGconcentration. If sufficient concentrations are reached in plasma andtissues to cause accumulation of GCase, the rate of accumulation will beat its maximum during the peaks (sometime after Cmax due to the lag timeassociated with penetration into the tissues, cells and ER), and at itsminimum during the troughs (some time after the Cmin). Thus the timebetween doses, Cmax and Cmin determine the net change in GCase levelsfor any given dosing interval.

Our model predicts that dosing regimens that maximize trafficking duringthe peaks and substrate turnover during troughs, will result in a sloweraccumulation rate of GCase than dosing regimens that favor traffickingduring both peaks and troughs. Therefore, for a given dose, the rate ofaccumulation of GCase will increase as the interval between doses isshortened and if the length of the dosing interval is held constant, therate of accumulation will also increase as the dose is increased (ifCmax<Emax). Taking this into consideration, our model predicts that wecould build-up enzyme levels in a relatively short period of time (1-2weeks) by administering a dosing regimen that favors traffickingthroughout the dosing interval, and maintain the elevated GCase levelsby switching to a regimen that provides peaks and troughs thatalternately maximize trafficking and substrate turnover.

Alternatively, initial “build-up” phases (several days long) could berepeated and separated by “drug free” phases (also several days long).

It should be noted that the foregoing was calculated based on theinteraction of IFG with the wild-type GCase enzyme. However, patientswith Gaucher disease will not have a wild-type enzyme, and thus the rateof enzyme turnover, residual level of enzyme activity, relative affinityof IFG for the enzyme, and dose that yields the maximum rate ofaccumulation will be different from that of the wild-type for eachmutation. For example, the most prevalent mutation in Gaucher disease isN370S. This mutant has a lower affinity for IFG compared to thewild-type, although the half-life is similar to that of the wild-type(Steet et al., PNAS 2006). Accordingly, the rate of N370S turnover andthe dose that will yield the maximum rate of GCase accumulation, can beestimated based on the differences in PK and PD parameters as comparedwith the wild-type enzyme:

TABLE 3 Parameter Wild-type N370S (expected) Max. rate of enzyme ~7Units/day ~0.35 Units/day accumulation Max. rate of enzyme loss ~−3.5Units/day ~−0.17 Units/day Residual level of enzyme ~20 Units ~1 Unitactivity Relative affinity for IFG 1 .33 Daily dose that yields max ≤75mg ≤150-300 mg rate of accumulation during Initial Enzyme Build-Up phase(1 unit = 1 nmole of 4-MU liberated per mg of total protein per hour)

From the foregoing, several dosing regimens for IFG to be administeredto Gaucher patients with the N370S mutation were modeled in silico(using the parameters discussed above). Specifically, the regimens wereas follows:

-   -   1. Two Different Maintenance Dosing Regimens Without an Initial        Enzyme Build-Up Phase        -   a. Administration of 150 mg of IFG every 3 days (FIG. 5A)        -   b. Administration of 150 mg of IFG every 4 days (FIG. 5A)    -   2. Initial Enzyme Build-Up Phase Followed by Two Different        Maintenance Dosing Regimens        -   a. Administration of 150 mg IFG daily for 7 days, followed            by administration of 150 mg of IFG every 4 days (FIG. 5B)        -   b. Administration of 150 mg of IFG daily for 7 days,            followed by administration of 150 mg of IFG every 7 days            (FIG. 5B)    -   3. Repeated Enzyme Build-Up Phases Separated by “Drug Free”        Phases        -   a. Administration of 150 mg of IFG daily for 4 days,            followed by 3 days without administration of IFG (FIG. 5C).        -   b. Administration of 150 mg of IFG daily for 3 days,            followed by 4 days without administration of IFG (FIG. 5C).

The results are presented in FIG. 5. The Dosing every 2 days without anInitial Enzyme Build-Up phase, is projected to result in an increase toa sustainable maximal GCase activity, but over a longer time. This maybe beneficial for people who have adverse side effects with dailyadministration of pharmacological chaperones and cannot tolerate anInitial Enzyme Build-Up phase.

The second regimen is expected to increase the rate of accumulation ofGCase in the lysosomes during the Initial Enzyme Build-Up Phase, whichis either maintained or gradually decreases over about 50 days duringthe Maintenance Phase. In this instance, subsequent Enzyme Build-UpPhases may need to be contemplated about every 35-40 days to permitre-accumulation of GCase in the lysosome, i.e., maximal chaperoning.

The third regimen is expected to increase the rate of GCase accumulationin the lysosome during repeated Enzyme Build-Up Phases (3-4 days), whilestill permitting dissociation of the chaperone and periods of maximalenzyme activity for substrate reduction during the intervening “drugfree” phase (3-4 days).

As one of skill in the art will appreciate, optimizing dose and dosinginterval for the treatment of patients with different mutations will bedetermined by specific properties of the mutant enzyme:

-   -   1. Half-life of the mutant enzyme: because some mutations result        in enzymes which may have shorter half-lives than N370S GCase,        shorter dosing intervals may be required for these mutants    -   2. Tissue half-life of chaperone: for chaperones with longer        tissue half-lives than plasma half-lives, a longer interval        between doses may be required    -   3. EC₅₀/IC₅₀: different mutant enzymes may have reduced affinity        for IFG, so the dose may need to be increased (adjust EC₅₀ and        IC₅₀ as needed).    -   4. Type of mutant: Many patients may have two different mutant        alleles (compound heterozygotes)—if both mutations are thought        to respond to IFG than a dosing regimen should be selected that        will be optimal for both mutations. Dose optimization should be        based on the highest EC₅₀ while the shortest half-life should be        taken into consideration when selecting a dosing interval or        duration of a Drug Free Phase. Additionally, priority for        optimization should be given to the mutant that is expected to        provide the greatest contribution to the total increase in GCase        activity.

Rationale Applied to Model Dosing Regimens for Fabry Disease

The model described above is readily applicable to estimating dosingregimens for other specific pharmacological chaperones for otherenzymes. As indicated earlier, the use of 1-deoxygalactonojrirmycinhydrochloride (DGJ) as a chaperone for α-galactosidse A (α-Gal A) forthe treatment of Fabry disease is being evaluated in clinical studies.

A multiple-dose Phase I trial was conducted and consisted of a total of16 healthy volunteers divided into two groups of eight subjects. Sixsubjects in each group received DGJ and two subjects received placebo.All subjects in one group received placebo or 50 mg twice a day forseven days, and all subjects in the other group received placebo or 150mg twice a day for seven days. Subjects were evaluated at the beginningof the study, on Day 7 after seven days of treatment and on Day 14 aftera seven day washout period.

The data from the multiple-dose Phase I trial showed a dose-relatedincrease in the level of α-GAL in the white blood cells of healthyvolunteers administered DGJ for seven days. At the highest dose levelthere was approximately a 2-fold increase in levels of α-GAL, and thisincrease was maintained for at least seven days after the last dose.

Results of in vitro and in vivo animal and Phase I studies using DGJ andwild-type α-GAL yielded the following PK and PD information (followingoral administration of 150 mg DGJ hydrochloride):

TABLE 4 Plasma PK and PD Cmax (μM) 9 Tmax (hrs) 3 Half-life (hrs;exponential decay) 3 Ki (μM) .04 EC₅₀ (μM) (cellular) 0.4 IC₅₀ (μM)(purified enzyme) 0.4

Since DGJ has a much shorter plasma half-life than IFG, the optimalMaintenance Dose is likely to be shorter than for IFG following theInitial Enzyme Build-Up Phase. As one example, it is predicted thatadministration of 150 mg DGJ every other day for 28 days will result ina plasma concentration above the EC₅₀ for about 16 hours on the day thedose is administered, and below the IC₅₀ for the remaining 8 hours (FIG.6). On the second day when no drug is administered, the plasmaconcentration is expected to be below the IC₅₀ until the following daywhen the drug is administered again (FIG. 6). This pattern continues forthe duration of the treatment period.

Specific Dosing Regimens for Gaucher Disease, Fabry Disease and PompeDisease

The following dosing regimens are specifically provided for Gaucher,Fabry and Pompe disease, but they can also be used for the treatment ofany lysosomal storage disorder that are amenable to treatment with thepharmacological chaperones described below.

Gaucher Disease.

In one embodiment of the invention, the Initial Enzyme Build-Up(loading) Phase, or the first phase of the dosing regimen, in which thepharmacological chaperone (e.g. IFG tartrate) is orally administereddaily will be from about 4 to about 10 days, or from about 5 to about 8days, or for about 7 days.

In this embodiment, the daily dose will be in a range from about 75 mgto about 300 mg per day, or from about 125 mg to about 225 mg, or about150 mg of pharmacological chaperone (e.g. IFG tartrate). Alternatively,a daily dose of 225 mg of pharmacological chaperone could beadministered.

Following completion of the first phase, the Maintenance Phase willbegin.

In one alternative embodiment, a first washout period will take placefollowing the first phase and prior to the maintenance phase.

In one embodiment, during the first washout period administration of thepharmacological chaperone from the first phase is stopped for a periodof between about 1 and 10 days, or from about 2 to 8 days, or about 7days.

In another embodiment, the first phase and the washout period can lastfor a period of about 1 week to about 30 weeks, or from about 2 weeks toabout 25 weeks, or about 2 weeks, or about 24 weeks.

In one embodiment, the interval for dosing during the Maintenance Phasewill be from about every 2 days to about every 8 days. In anotherembodiment, the interval will be from about every 4 days to about every7 days. In a third embodiment, the interval will be about every 7 days.

In these embodiments, the dose administered during the Maintenance Phase(the “Maintenance Dose”) will be in a range from about 75 mg to about300 mg, or, in one aspect, about 150 mg per dose of pharmacologicalchaperone (e.g. IFG tartrate), or in another aspect 225 mg per dose.These dosages are administered once per interval described above

Alternatively, the maintenance phase may consist of daily administrationfor a period of time. In one embodiment the maintenance phase period canbe between 1 and 8 days, or about 4 and 7 days, or about three days, orabout 7 day, followed by a second “washout period” of substantiallyequal duration. For example the maintenance phase may consist of 3 dailydosages of pharmacological chaperone (e.g. IFG tartrate) from about 75mg to about 300 mg, or from about 125 mg to about 275 mg, or 225 mgfollowed by between about 1 and 10 days, or about 2 and 8 days, or aboutfour days of washout, i.e. without any pharmacological chaperoneadministered.

In a further embodiment, the maintenance phase and the washout periodcan last for a period of between about 1 week and about 30 weeks, orbetween about 2 weeks and about 25 weeks, or about 22 weeks.

In another alternative embodiment, there can be no maintenance phase andsecond washout period.

In another embodiment, the patient does not ingest any food (i.e.“fasts”) prior to and following the administration of a pharmacologicalchaperone for a period of between about 0.5 and about 24 hours, orbetween about 1 and about 12 hours, or about 2 hours.

In one specific example, 150 mg of IFG tartrate is administered dailyfor seven days (Enzyme Build-up Phase). After these seven days 225 mg ofIFG tartrate is administered daily for 3 days (3 days on) followed by awashout period of 4 days (4 days off). The 3 days on/4 days off regimenis repeated indefinitely

In another specific example, 150 mg of IFG tartrate is administereddaily for seven days. After these seven days 225 mg is administereddaily for 7 days (7 days on) followed by a washout period of 7 days (7days off). The 7 days on/7 days off regimen is repeated indefinitely.

In another specific example, 225 mg of IFG tartrate is administereddaily for seven days. (7 days on) followed by a washout period of 7 days(7 days off). The 7 days on/7 days off regimen is repeated for a periodof 24 weeks.

In another specific example, 225 mg of IFG tartrate is administereddaily for seven days. (7 days on) followed by a washout period of 7 days(7 days off). Next, 225 mg of IFG tartrate is administered daily for 3days (3 days on) during a maintenance phase, followed by a secondwashout period of 4 days (4 days off). The 3 days on/4 days off regimenis repeated for a period of 22 weeks.

In another aspect of the invention, sustained low plasma concentrationsmay be desirable following the dose administered during the InitialEnzyme Build-Up Phase. In this embodiment, an Initial Enzyme Build-UpPhase is envisioned at a dose capable of resulting in maximum increasesin enzyme level, followed by a much lower daily dose for a MaintenanceDose that is capable of sustaining an increased level of enzyme exitingthe ER while also permitting dissociation of the chaperone once theenzyme is in the lysosome.

In this embodiment, the Initial Enzyme Build-Up Phase, or the firstphase of the dosing regimen, in which the pharmacological chaperone(e.g. IFG tartrate) is orally administered daily will be from about 4 toabout 14 days, or from about 5 to about 10 days, or for about 7 days,and the loading dose will be in a range from about 75 mg to about 300 mgper day, or from about 125 mg to about 225 mg, or about 150 mg.

Following completion of the first phase, the Maintenance Phase willbegin in which the daily dose is reduced to about 25 to 50 mg, or about25 mg of pharmacological chaperone (e.g. IFG tartrate).

In a third aspect of the invention, interval dosing about every 2-3 daysis contemplated. In this embodiment, between about 75 mg to about 300 mgof pharmacological chaperone (e.g. IFG tartrate) is administered at eachinterval, or from about 125 mg to about 225 mg at each interval, orabout 150 mg at each interval.

For all of the foregoing embodiments, if the interval during theMaintenance Phase is 3 days instead of two days, it may be moreefficacious to administer higher doses.

Alternatively, the dosing regimen may consist of administration of aconstant amount of pharmacological chaperone over a specific timeperiod. For example a constant amount of pharmacological chaperone maybe administered twice daily, once daily, once every 3 days, once every 4days, once every week, once every two weeks, or once a month. This cyclemay be repeated indefinitely.

In one embodiment, from about 10 mg to about 200 mg of pharmacologicalchaperone (e.g. IFG tartrate) is administered daily. For example, 10 mg,or 25 mg, or 50 mg, or 75 mg, or 100 mg or 125 mg or 150 mg, or 175 mg,or 200 mg of pharmacological chaperone (e.g. IFG tartrate) isadministered daily.

In one specific embodiment, 25 mg/day of IFG tartrate is administered.In another embodiment, 150 mg/day of IFG tartrate is administered.

In an alternative embodiment, from about 10 mg to about 400 mg ofpharmacological chaperone (e.g. IFG tartrate) is administered once everythree days, once every four days, or alternatively once every week. Forexample, 10 mg, or 25 mg, or 50 mg, or 75 mg, or 100 mg or 125 mg or 150mg, or 175 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg ofpharmacological chaperone (e.g. IFG tartrate) is administered once everythree days, once every four days, or once every week.

In one specific embodiment, 150 mg of IFG tartrate is orallyadministered every four days. In another embodiment, 150 mg/day of IFGtartrate is orally administered once every week.

Fabry Disease.

In one embodiment of the invention, the Initial Enzyme Build-Up(loading) Phase, or the first phase of the dosing regimen, in which apharmacological chaperone (e.g. DGJ hydrochloride) is orallyadministered daily will be from about 4 to about 10 days, or from about5 to about 8 days, or for about 7 days.

In this embodiment, the daily dose of pharmacological chaperone (e.g.DGJ hydrochloride) during the first phase will be in a range from about200 mg to about 500 mg per day, or from about 250 mg to about 300 mg perday, or about 250 mg per day. These levels may be achieved gradually inan ascending manner. For example, the dose of pharmacological chaperone(e.g. DGJ hydrochloride) may begin at 25 mg for a period of time (e.g. 2weeks), then progressed to 100 mg for a period of time (e.g. two weeks),and then progressed to the highest dose administered for the remainderof the Build-Up Phase.

Alternatively, the maximum amount administered during the build-up phasemay be administered initially, i.e. no ascending dosages.

Following completion of the first phase, the Maintenance Phase willbegin. In one embodiment, the interval for dosing during the MaintenancePhase will be from about every 2 days to about every 3 days. In anotherembodiment, the interval will be from about every 2 days.

In these embodiments, the Maintenance Dose will be in a range from about75 mg to about 225 mg per dose, or, from about 100 mg to about 200 mgper dose, or, in a specific embodiment, about 150 mg per dose.

In another embodiment, sustained low steady-state plasma concentrationsmay be desirable following the Initial Enzyme Build-Up Phase. In thisembodiment, a Initial Enzyme Build-Up Phase is envisioned at a dosecapable of resulting in maximum increases in enzyme level, followed by amuch lower daily dose that is capable of sustaining an increased levelof enzyme exiting the ER while also permitting dissociation of thechaperone once the enzyme is in the lysosome.

In this embodiment, Initial Enzyme Build-Up Phase, or the first phase ofthe dosing regimen, in which the pharmacological chaperone (e.g. DGJhydrochloride) is orally administered daily will be from about 4 toabout 14 days, or from about 5 to about 10 days, or in a particularembodiment, for about 7 days, and the loading dose of pharmacologicalchaperone (e.g. DGJ hydrochloride) may be in a range from about 200 mgto about 500 mg per day, or from about 250 mg to about 300 mg per day,or about 250 mg per day.

Alternatively, the first phase may last for a period from about twoweeks to about 12 weeks, for from about 4 weeks to about 8 weeks (e.g. 6weeks). The loading dose of pharmacological chaperone (e.g. DGJhydrochloride) may be in a range from about 200 mg to about 500 mg perday, or from about 250 mg to about 300 mg per day, or about 250 mg perday.

As noted above, the these dosage amounts during the Build-Up Phase maybe achieved in an ascending manner, or the maximum amount administeredduring the build-up phase may be administered initially.

Following completion of the first phase, a reduction in the daily dosewill begin. In this embodiment, the daily dose is reduced to about 25 to50 mg, or about 25 mg. This is the Maintenance Dose.

In one specific embodiment, the Build-Up Phase consists of 2 weeks at 25mg/day, 2 weeks at 100 mg/day and 2 weeks at 250 mg/day of orallyadministered DGJ hydrochloride, followed by a period of time (e.g. 24weeks) at 25 mg/day, followed by a period of time (e.g. 66 weeks) of 50mg/day or orally administered DGJ hydrochloride.

In a third aspect of the invention, interval dosing about every 2-3 daysis contemplated. In this embodiment, between about 25 mg to about 300 mgof pharmacological chaperone (e.g. DGJ hydrochloride) is administered ateach interval, or from about 125 mg to about 225 mg at each interval, orabout 150 mg at each interval. In specific embodiments, DGJhydrochloride will be administered at 50 mg, 150 mg or 250 mg every 2days.

For all of the foregoing embodiments, if the interval is 3 days insteadof two days, it may be more efficacious to administer higher intervaldoses.

Similar as for IFG described above, if a patient cannot tolerate thedose administered during the Initial Enzyme Build-Up Phase, and intervaldosing without this phase will not achieve plasma concentrations at orabove the EC₅₀, a more gradual “loading” period, followed by a low dailyMaintenance Dose may be appropriate. For example, in one embodiment, DGJwill be administered 50 mg per day for two weeks, followed by 200 mg perday for two weeks, followed by 500 mg per day for two weeks and followedby 50 mg per day for the duration of treatment.

In various embodiments, during the Enzyme Build-Up Phase, the dosage mayescalate upwards in dosage amount. For example, ascending dosages of 25,100, and 250 mg may be administered for one day each, i.e. 25 mg on day1, 100 mg on day 2, and 250 mg on day 3. Such embodiments may used toslowly acclimate the subject to higher dosage amounts during the EnzymeBuild-Up Phase. Alternatively, the dosage amount during the EnzymeBuild-Up Phase may be constant throughout the duration of the phase.

In one embodiment, from about 75 mg to about 800 mg per day ofpharmacological chaperone, or from about 125 mg to about 600 mg, or 250mg or 500 mg is administered once a day for a period of one to sevendays, followed by a washout period of equal or substantially equalduration. For example, the dosing regimen may consist of threeconsecutive days of receiving a daily dosage of pharmacologicalchaperone (e.g. DGJ or DGJ hydrochloride), followed by four consecutivedays of not receiving the dosage; or four consecutive days of receivinga daily dosage, followed by three consecutive days of not receiving thedosage.

In one specific example, a dosing regimen for Fabry Disease may call fororal administration of 250 mg, or 500 mg of DGJ hydrochloride once a dayfor three consecutive days, followed by four days without taking apharmacological chaperone (i.e. DGJ hydrochloride). Alternatively, thedosing regimen may consist of 250 or 500 mg of pharmacological chaperone(e.g. DGJ or DGJ hydrochloride) once a day for seven consecutive days,followed by seven days without taking the pharmacological chaperone.

Alternatively, from about 75 mg to about 300 mg per day, or from about125 mg to about 225 mg, or 150 mg may be administered for one to sevendays followed by a washout period of unequal duration. For example, thedosing regimen may call for one day of receiving a dosage, followed bysix consecutive days of not receiving the dosage; two consecutive daysof receiving a daily dosage, followed by five consecutive days of notreceiving the dosage; five consecutive days of receiving a daily dosage,followed by two consecutive days of not receiving the dosage; or sixconsecutive days receiving a daily dosage, followed by one day of notreceiving the dosage.

Pompe Disease.

In one embodiment, from about 1000 mg to about 8000 mg per day ofpharmacological chaperone (e.g. DNJ), or from about 2000 mg to about6000 mg, or 2500 mg or 5000 mg of pharmacological chaperone is orallyadministered once a day for a period of one to seven days, followed by awashout period of equal or substantially equal duration. For example,the dosing regimen may consist of 3 or 4 days “on” (daily administrationof the pharmacological chaperone), followed by 4 or 3 days “off” (notadministering the pharmacological chaperone). Alternatively, the dosingregimen may consist of seven days on and seven days off.

In one specific embodiment, 2500 mg of DNJ (including pharmaceuticallyacceptable salts thereof) is orally administered daily for threeconsecutive days, followed by four consecutive days of not administeringa pharmacological chaperone. In an alternative embodiment, 5000 mg ofDNJ (including pharmaceutically acceptable salts thereof) is orallyadministered daily for three consecutive days, followed by fourconsecutive days of not administering a pharmacological chaperone. In analternative embodiment, 5000 mg of DNJ (including pharmaceuticallyacceptable salts thereof) is orally administered daily for sevenconsecutive days, followed by seven consecutive days of notadministering a pharmacological chaperone.

The above dosage amounts may be achieved in an ascending fashion. Forexample, the dosage amount in the first cycle (i.e. the first three daysor first seven days) may be 500 mg, 1000 mg during the second cycle,2500 mg during the third cycle and 5000 during the fourth cycle.

A person of ordinary skill in the art will be able to apply thisstrategy to estimate appropriate dosing regimens for otherpharmacological chaperones which are competitive inhibitors of lysosomalenzymes to treat other lysosomal storage diseases, based on the specificPK and PD for each enzyme and candidate chaperone.

Formulation and Administration

Isofagomine can be administered in a form suitable for any route ofadministration, including e.g., orally in the form tablets, capsules, orliquid, or in sterile aqueous solution for injection. It can beadministered orally in the form of tablets, capsules, ovules, elixirs,solutions or suspensions, gels, syrups, mouth washes, or a dry powderfor constitution with water or other suitable vehicle before use,optionally with flavoring and coloring agents for immediate-, delayed-,modified-, sustained-, pulsed-or controlled-release applications. Solidcompositions such as tablets, capsules, lozenges, pastilles, pills,boluses, powder, pastes, granules, bullets, or premix preparations mayalso be used. Solid and liquid compositions for oral use may be preparedaccording to methods well known in the art. Such compositions may alsocontain one or more pharmaceutically acceptable carriers and excipientswhich may be in solid or liquid form. When the compound is formulatedfor oral administration, the tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart.

The pharmaceutically acceptable excipients also include microcrystallinecellulose, lactose, sodium citrate, calcium carbonate, dibasic calciumphosphate and glycine, disintegrants such as starch (preferably corn,potato or tapioca starch), sodium starch glycolate, croscarmellosesodium and certain complex silicates, and granulation binders such aspolyvinylpyrolidone, hydroxypropyl ethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar, or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the agent may becombined with various emulsifying and/or suspending agents and withdiluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for constitution with water or another suitable vehicle (forexample, ethanol or a polyol such as glycerol, propylene glycol, andpolyethylene glycol, and the like, suitable mixtures thereof, andvegetable oils) before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., water, sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). Preparations for oraladministration may be suitably formulated to give controlled orsustained release of the ceramide-specific glucosyltransferaseinhibitor.

The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, benzyl alcohol, sorbic acid, and the like. Inmany cases, it will be reasonable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate, and gelatin.

The pharmaceutical formulations of isofagomine suitable forparenteral/injectable (for example, by intravenous bolus injection orinfusion or via intramuscular, subcutaneous or intrathecal routes) usegenerally include sterile aqueous solutions, or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The isofagomine tartrate may be presented inunit dose form, in ampoules, or other unit-dose containers, or inmulti-dose containers, if necessary with an added preservative. Thecompositions for injection may be in the form of suspensions, solutions,or emulsions, in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, solubilizing, and/or dispersingagents. Alternatively, the active ingredient may be in sterile powderform for reconstitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use. In all cases, the form must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art.

Sterile injectable solutions are prepared by incorporating isofagominein the required amount in the appropriate solvent with various of theother ingredients enumerated above, as required, followed by filter orterminal sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

Preservatives, stabilizers, dyes, and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, ascorbic acid, and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Additional pharmaceutically acceptable carriers which may be included inthe formulation are buffers such as citrate buffer, phosphate buffer,acetate buffer, and bicarbonate buffer, amino acids, urea, alcohols,ascorbic acid, phospholipids, proteins, such as serum albumin, collagen,and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomespolyvinylpyrolidone; sugars such as dextran, mannitol, sorbitol, andglycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000,PEG-6000); glycerol, glycine or other amino acids and lipids. Buffersystems for use with the formulations include citrate, acetate,bicarbonate, and phosphate buffers. Phosphate buffer is a preferredembodiment.

The formulations can also contain a non-ionic detergent. Preferrednon-ionic detergents include Polysorbate 20, Polysorbate 80, TritonX-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octyl β-glucoside,Brij 35, Pluronic, and Tween 20.

The routes for administration (delivery) include, but are not limitedto, one or more of: oral (e.g., as a tablet, capsule, or as aningestible solution), topical, mucosal (e.g., as a nasal spray oraerosol for inhalation), nasal, parenteral (e.g., by an injectableform), gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intrauterine, intraocular, intradermal, intracranial,intratracheal, intravaginal, intracerebroventricular, intracerebral,subcutaneous, ophthalmic (including intravitreal or intracameral),transdermal, rectal, buccal, epidural and sublingual.

Administration of the above-described parenteral formulations ofisofagomine may be by periodic injections of a bolus of the preparation,or may be administered by intravenous or intraperitoneal administrationfrom a reservoir which is external (e.g., an i.v. bag) or internal(e.g., a bioerodable implant). See, e.g., U.S. Pat. Nos. 4,407,957 and5,798,113, each incorporated herein by reference. Intrapulmonarydelivery methods and apparatus are described, for example, in U.S. Pat.Nos. 5,654,007, 5,780,014 and 5,814,607, each incorporated herein byreference. Other useful parenteral delivery systems includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, pump delivery, encapsulated cell delivery, liposomaldelivery, needle-delivered injection, needle-less injection, nebulizer,aeorosolizer, electroporation, and transdermal patch. Needle-lessinjector devices are described in U.S. Pat. Nos. 5,879,327, 5,520,639,5,846,233 and 5,704,911, the specifications of which are hereinincorporated by reference. Any of the formulations described above canbe administered using these methods. Furthermore, a variety of devicesdesigned for patient convenience, such as refillable injection pens andneedle-less injection devices, may be used with the formulations of thepresent invention as discussed herein.

In a specific embodiment, isofagomine tartrate is administered as apowder-filled capsule, with lactose and magnesium stearate asexcipients.

Combination Therapy.

The pharmaceutical composition may also include other biologicallyactive substances in combination with the candidate compound(pharmacological chaperone) or may be administered in combination withother biologically active substances. Such combination therapy includes,but is not limited to, combinations with replacement enzymes such asCerezyme®, Fabrazyme®, Aldurazyme®, Myozyme® and Replagal®; combinationswith substrate reduction therapies (also known as substrate depravationtherapy), such as Zavesca® or those molecules disclosed, for example, inU.S. Pat. Nos. 6,916,802 and 6,051,598, hereby incorporated byreference; and combinations with gene therapy vectors or cellscontaining a gene for GCase.

EXAMPLES Example 1: Dosing Regimen for the Treatment of Gaucher DiseaseUsing Isofagomine Tartrate

The primary objective of the study is to evaluate the safety,tolerability and pharmacodynamics of two dose regimens of orallyadministered IFG tartrate in patients with type 1 GD. As indicatedabove, the prevalent mutation in Type 1 GD is N370S.

This will be a Phase 2, randomized, two dose group, open-label study toassess the safety and tolerability of isofagomine tartrate. The studywill be conducted in treatment-naïve patients with type 1 GD between theages of 18 and 65 years. Approximately 16 subjects will be enrolled.

This study will consist of a 7-day screening period, followed byrandomization for qualifying subjects, a 24-week treatment period, whichwill be followed by a 14-day follow-up period.

Visits are scheduled at Day −7 (±3 days), Day 1 (±3 days), Day 7 (±3days), Day 14 (±3 days), Day 28 (±3 days), Day 56 (±3 days), Day 84 (±3days), Day 112 (±3 days), Day 140 (±3 days), Day 168 (±3 days) and Day182 (±3 days). If a subject is withdrawn from the study after Day 1 andprior to study completion, the subject will be encouraged to undergo allprocedures scheduled at Day 168 (visit 10).

At Day 1, subjects will be randomized in equal proportions to one of thetwo following groups:

-   -   1. Isofagomine tartrate, 150 mg orally every day for 1 week        followed by 150 mg every 4 days for 23 weeks    -   2. Isofagomine tartrate, 150 mg orally every day for 1 week        followed by 150 mg every 7 days for 23 weeks

IFG tartrate is administered in 25 mg capsules. Since a food effect isanticipated, patients will have no food for two hours prior and twohours following drug administration.

The secondary objective of the study is to assess pharmacodynamiceffects of two dose groups of orally administered isofagomine tartratein treatment-naïve patients with type 1 Gaucher disease. Secondaryendpoints which will be evaluated are as follows:

-   -   β-glucocerebrosidase (GCase) levels in white blood cells    -   Glucocerebroside (GlcCer) levels in white blood cells    -   α-synuclein levels in plasma    -   Bone-specific alkaline phosphatase activity in plasma (BAP)        activity in plasma    -   Chitotriosidase activity in plasma    -   Interleukin 8 levels in plasma    -   Interleukin 17 levels in plasma    -   Macrophage Inflammatory Protein 1a (MIP-1a) level in plasma    -   Pulmonary and activation regulated chemokine (PARC) activity in        plasma    -   Tartrate-resistant acid phosphatase 5b (TRACP 5b) activity in        plasma    -   Vascular Endothelial Growth Factor (VEGF) levels in plasma    -   Change in liver volume from baseline    -   Change in spleen volume from baseline    -   Change in hemoglobin level from baseline    -   Change in hematocrit level from baseline    -   Change in platelet count from baseline    -   Change in bone mineral density of left or right femoral bones        from baseline    -   Change in bone mineral density of the spine from baseline    -   Change in radiographic findings of the left or right femoral        bones from baseline    -   Change in radiographic findings of spine from baseline

The post-baseline pharmacodynamics parameters will be compared withbaseline values by a two-tailed paired t-test procedure at the 95%confidence level. A repeated measure analysis of variance model will beinvoked to determine the treatment effects on the values ofpharmacodynamic parameters. In this analysis model, genotype and subjectwithin genotype will be random effects, treatment, visit andvisit-by-treatment interaction will be fixed effects and baseline valuewill be the covariate. An autoregressive model will be used to model thecovariance structure among different time points. The treatmentcomparison will be assessed at the 5% significance level. In addition,the 95% confidence interval for the treatment difference will beprovided.

It is anticipated that one or both of these loading/interval dosingregimens using IFG will be therapeutically effective for the treatmentof Gaucher disease. Some genotypes anticipated to respond to this dosingregimen include but are not limited to the following: N370S/N370S,N370S/L444P, N370S/84insG, N370S/R163X, N370S/Y212H, L444P/del 136T,L444P/F216Y, L444P/L174F, G202R/R463C, L444P/L444P, and K79N/complex Bexon 9/10 (type 3 GD).

Example 2: Dosing Regimen for the Treatment of Gaucher Disease UsingIsofagomine Tartrate

The primary objective of the study is to evaluate the safety,tolerability and pharmacodynamics of one dose regimen of orallyadministered IFG tartrate in patients with type 1 GD.

This will be a Phase 2, randomized, two dose group, open-label study toassess the safety and tolerability of isofagomine tartrate. The studywill be conducted in patients with type 1 GD between the ages of 18 and65 years. Approximately 16 subjects will be enrolled.

Visits are scheduled at Day −7 (±3 days), Day 1 (±3 days), Day 7 (±3days), Day 14 (±3 days), Day 28 (±3 days), Day 56 (±3 days), Day 84 (±3days), Day 112 (±3 days), Day 140 (±3 days), Day 168 (±3 days) and Day182 (±3 days). If a subject is withdrawn from the study after Day 1 andprior to study completion, the subject will be encouraged to undergo allprocedures scheduled at Day 168 (visit 10).

This study will consist of a 7-day screening period, followed byrandomization for qualifying subjects, a 24-week treatment period, whichwill be followed by a 14-day follow-up period.

At Day 1, subjects will be randomized in equal proportions to placebo orIsofagomine tartrate, 150 mg orally every 3 days for the entiretreatment period. IFG tartrate is administered in 25 mg capsules. Sincea food effect is anticipated, patients will fast for two hours prior andtwo hours following drug administration.

Evaluation of secondary objectives will be performed as outlined abovefor Example 1.

It is anticipated that this dosing regime will be therapeuticallyeffective for the treatment of Gaucher disease.

Example 3: Administration of Single Dose DGJ to Evaluate Safety,Tolerability and Pharmacokinetics, and Affect on α-Galatosidase aEnzymatic Activity

This example describes a randomized, double blind, placebo controlledPhase Ib study of twice daily oral doses of DGJ to evaluate the affectsof DGJ on safety, tolerability, pharmacokinetics, and α-Galatosidase A(α-GAL) enzymantic activity in healthy volunteers.

Study Design and Duration.

This study was first-in-man, single-center, Phase Ib, randomized,double-blind, twice daily-dose, placebo controlled study to evaluate thesafety, tolerability, pharmacokinetics, and α-GAL enzymantic activityaffects of DGJ following oral administration. The study tested twogroups of 8 subjects (6 active and 2 placebo) who received a twicedaily-dose of 50 or 150 mg b.i.d. of DGJ or placebo administered orallyfor seven consecutive days, accompanied by a seven day follow up visit.Subjects were housed in the treatment facility from 14 hours prior todosing until 24 hours after dosing. Meals were controlled by scheduleand subjects remained abulatory for 4 hours post drug administration

Pharmacokinetic parameters were calculated for DGJ in plasma on Day 1and Day 7. In addition, the cumulative percentage of DGJ excreted (12hours post dose) in urine was calculated. α-GAL activity was calculatedin white blood cells (WBC) before dosing began, and again at 100 hours,150 hours, and 336 hours into the trial.

Study Population.

Subjects were healthy, non-institutionalized, non-smoking malevolunteers between 19 and 50 years of age (inclusive) consisting ofmembers of the community at large.

Safety and Tolerability Assessments.

Safety was determined by evaluating vital signs, laboratory parameters(serum chemistry, hematology, and urinalysis), ECGs, physicalexamination and by recording adverse events during the Treatment Period.

Pharmacokinetic Sampling.

Blood samples (10 mL each) were collected in blood collection tubescontaining EDTA before dosing and at the following times thereafter:0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 hours.Blood samples were cooled in an ice bath and centrifuged underrefrigeration as soon as possible. Plasma samples were divided into twoaliquots and stored at 20±10° C. pending assay. At the end of the study,all samples were transferred to MDS Pharma Services AnalyticalLaboratories (Lincoln) for analysis. The complete urine output wascollected from each subject for analysis of DGJ to determine renalclearance for the first 12 hours after administration of DGJ on days 1and 7.

WBC α-GAL Enzymatic Activity Sampling.

Blood samples (10 mL each) were collected in blood collection tubescontaining EDTA and WBC extracted before dosing and at the followingtimes thereafter: 100 hours, 150 hours, and 336 hours. Samples weretreated as described above, and WBC α-GAL enzymatic activity levels weredetermined as described in Desnick, R. J. (ed) Enzyme therapy in geneticdiseases. Vol 2. Alan R Liss, New York, pp 17-32. Statistical Analysis.Safety data including laboratory evaluations, physical exams, adverseevents, ECG monitoring and vital signs assessments were summarized bytreatment group and point of time of collection. Descriptive statistics(arithmetic mean, standard deviation, median, minimum and maximum) werecalculated for quantitative safety data as well as for the difference tobaseline. Frequency counts were compiled for classification ofqualitative safety data. In addition, a shift table describing out ofnormal range shifts was provided for clinical laboratory results. Anormal-abnormal shift table was also presented for physical exam resultsand ECGs.

Adverse events were coded using the MedDRA version 7.0 dictionary andsummarized by treatment for the number of subjects reporting the adverseevent and the number of adverse events reported. A by-subject adverseevent data listing including verbatim term, coded term, treatment group,severity, and relationship to treatment was provided. Concomitantmedications and medical history were listed by treatment.

Pharmacokinetic parameters were summarized by treatment group usingdescriptive statistics (arithmetic means, standard deviations,coefficients of variation, sample size, minimum, maximum and median).

Results

No placebo-treated subjects had AEs and no subject presented with AEsafter receiving 50 mg b.i.d. or 150 mg b.i.d. DGJ. DGJ appeared to besafe and well tolerated by this group of healthy male subjects as doseswere administered at 50 mg b.i.d. and 150 mg b.i.d.

Laboratory deviations from normal ranges occurred after dosing, but nonewas judged clinically significant. There were no clinically relevantmean data shifts in any parameter investigated throughout the course ofthe study. No clinically relevant abnormality occurred in any vitalsign, ECG, or physical examination parameter.

Pharmacokinetic Evaluation.

The following table summarizes the pharmacokinetic data obtained duringthe study.

TABLE 5 50 mg bid dose 150 mg bid dose Day 1 Day 7 Day 1 Day 7 Cmax (μM)2.3 ± 0.3 3.9 ± 0.5 11.3 ± 1.5 10.8 ± 1.4  tmax (h) 2.9 ± 0.4 2.5 ± 0.4 3.1 ± 0.4 2.9 ± 0.4 t½ (h) 2.5 ± 0.1  2.4 ± 0.05 Cmin (μM)  0.4 ± 0.031.2 ± 0.1 12 h cumulative 16 ± 6  48 ± 7  42 ± 7 60 ± 5  renal excretion(%)^(a) ^(a)Cumulative percentage of DGJ excreted over the 12-hour postdose period.

The pharmacokinetics of DGJ were well characterized in all subjects andat all dose levels. On average, peak concentrations occurred atapproximately 3 hours for all dose levels. C_(max) of DGJ increased in adose-proportional manner when doses were increased from 50 mg to 150 mg.

The mean elimination half-lives (t_(1/2)) were comparable at dose levelsof 50 and 150 mg on Day 1 (2.5 vs. 2.4 hours).

The mean percentage of DGJ excreted over the 12-hour post dose periodwas 16% and 42% at dose levels of 50 and 150 mg, respectively, on Day 1,increasing to 48% and 60%, respectively, on Day 7.

α-Galactosidase A (α-GAL) Enzymatic Activity.

The α-GAL enzymatic activity data obtained during the study is shown inFIG. 1. DGJ did not inhibit WBC α-GAL enzymatic activity in subjects atdosages of 50 mg b.i.d. or 150 mg b.i.d. Furthermore, DGJ produced adose-dependent trend of increased WBC α-GAL activity in healthyvolunteers. α-GAL enzymatic levels were measured in WBC of subjectsadministered placebo, 50 mg b.i.d. DGJ, and 150 mg b.i.d. DGJ. Placebohad no affect on WBC α-GAL enzymatic levels. Variations in enzymaticlevels in response to placebo were not clinically significant. Both 50mg b.i.d. and 150 mg b.i.d. DGJ increased normalized WBC α-GAL enzymaticlevels. In response to 50 mg b.i.d. DGJ, WBC α-GAL enzymatic activityincreased to 120%, 130%, and 145% pre-dose levels at 100 hours, 150hours, and 336 hours post-dose, respectively. In response to 150 mgb.i.d. DGJ, WBC α-GAL enzymatic activity increased to 150%, 185%, and185% pre-dose levels at 100 hours, 150 hours, and 336 hours post-dose,respectively.

Example 4: Dosing Regimen for the Treatment of Fabry Disease Using DGJHydrochloride

This example describes a dosing regiment using DGJ that is contemplatedfor the treatment of Fabry patients.

Patient Enrollment.

Fabry patients with known missense mutations in α-GAL (verified bygenotype); patients currently receiving ERT (Fabrazyme®) who are willingto stop ERT for up to 6 months; or newly diagnosed patients who havenever been treated with ERT.

Study Design.

Patients will be orally administered DGJ hydrochloride or a placebodaily for 7 days at a dose of 250 mg/day. This is the Initial EnzymeBuild-Up Phase. Following completion of the first phase, the MaintenancePhase will begin wherein DGJ or a placebo is administered at aMaintenance dose of 150 mg every other day.

GL-3 deposits. Skin, kidney and heart biopsies will be performed atbaseline, 3 months and six months and evaluated for GL-3 deposits inskin fibroblasts, cardiac myocytes, and various renal cells. It isanticipated that clearance of GL-3 will be observed in all cells.Clearance in cardiac myocytes or renal podocytes or skin tissue has notpreviously been shown upon treatment with ERT (although changes inurinary sediment at 6 and 18 months of ERT suggested that accumulationsof glycosphingolipids in renal tissues were cleared by enzymereplacement; Clin Chim Acta. 2005; 360(1-2): 103-7).

α-GAL activity. In addition, α-GAL activity will be assessed infractionated tissue obtained from biopsies and in blood leukocytes andplasma (from blood collected at baseline and every month). It isanticipated that DGJ treatment as monotherapy and in combination withERT will increase α-GAL activity from about 2-fold to 10-fold abovebaseline in leukocytes, fibroblasts and plasma. It is also anticipatedthat increases in α-GAL activity will be observed in tissue, which hasnot been demonstrated with ERT treatment.

Urinalysis.

Urine and urinary sediment will be analysed at baseline and monthly forα-GAL and GL-3. In addition, the abnormal presence of other lipids, suchas CTH, lactosylceramide, ceramide, and abnormal decrease or absence ofglucosylceramide and sphingomyelin will also be evaluated

Urine will also be analyzed for the presence of protein includingalbumin (proteinuria) and creatine to monitor the status of renaldisease.

It is anticipated that DGJ treatment will reduce proteinurea and reduceGL-3 sedimient.

Cardiac Anaylsis.

In addition to the biopsies described above, MRIs and echocardiogramwith strain rate evaluations will be performed at baseline, 3 and 6months to assess cardiac morphology (e.g., left ventricular hyertrophy)and cardiac function (e.g., congestive heart failure, ischemia,infarction, arrhythmia) Direct reduction in left ventricularhypertrophy, or increase in left ventricular ejection fraction, hasnever been demonstrated by other treatments. Hypertension will also beevaluated since hypertension (associated with renal dysfunction) canincrease the risk for hemorrhagic stroke.

Electrocardiograms will be performed at baseline and at every visit foranalysis of improvement in any conduction abnormalities, arrhythmias,bundle branch blocks, or tachy or bradycardia. Previous treatments havenot shown improvements in patients presenting with these symptoms.

Renal Analysis.

Renal podocytes will be evaluated using light and electron microscopyfor clearance of GL-3.

Brain Analysis.

MRI and MRA will be performed at baseline and at the end of the study toassess for a reduction in ischemic arease, which can cause ischemicstrokes. The reduction in GL-3 buildup by DGJ is anticipated to reducethe incidence of strokes. Since replacement enzyme cannot cross theblood brain-barrier, improvements in brain ischemia has never beenachieved with ERT.

Opthamology.

Ophthalmologic exams will be performed to assess reduction in cornealand lens opacities such as cataracts.

Neuropathic Pain.

Subjective patient questionnaires will be administered to patients atbaseline and at each monthly visit to evaluate reduction inacroparaesthesia. This may be evidence of clearance of GL-3 in themicrovasculature of peripheral nerve cells.

Neuropathy.

Quantitative Sensory Testing (CASE study) will be used to evaluateperipheral neuropathy.

Hypohidrosis.

Sweat glands will be evaluated using quantitative sudomotor axon reflextest (QSART), which assesses the small nerve fiblers that are linked tothe eccrine sweat glands. Improvements in the sweat glands shouldcorrelate with an increase in sweating, and may also be evidence ofclearance of GL-3 in the microvasculature of peripheral nerve cells.This analysis will be performed at baseline and at 3 and 6 months.

It is anticipated that this dosing regime will be therapeuticallyeffective for the treatment of Fabry disease. Some specific missensemutations expected to respond to treatment with DGJ include, but are notlimited to, L32P, N34S, T41L, M51K, E59K, E66Q, I91T, A97V, R100K,R112C, R112H, F113L, G132R, A143T, G144V, S148N, D170V, C172Y, G183D,P205T, Y207S, Y207C, N215S, R227X, R227Q, A228P, S235C, D244N, P259R,N263S, N264A, G271C, S276G, Q279E, M284T, W287C, I289F, F295C, M296I,M296V, L300P, R301Q, V316E, N320Y, G325D, G328A, R342Q, R356W, E358A,E358K, R363C, R363H, and P409A.

Example 5: Dosing Regimens for the Treatment of Fabry Disease Using DGJHydrochloride

This example describes a Phase II study of DGJ in Fabry patients.Patient enrollment. Fabry patients with known missense mutations inα-GAL (verified by genotype); patients currently receiving ERT(Fabrazyme®) who are willing to stop ERT for up to 6 months; or newlydiagnosed patients who have never been treated with ERT.

Study Design.

Eight patients in the study received an ascending dose of 25, 100, andthen 250 mg b.i.d. over 6 weeks, followed by 50 mg/day for the remainderof the study. Three patients in the study received 150 mg of DGJ everyother day throughout the entire study.

Some of the same surrogate markers as described for Example 4 will bemonitored during the study.

Results

α-GAL Activity.

The available data from the first eleven patients treated with DGJ forat least 12 weeks suggest that treatment with DGJ leads to an increasein the activity of the enzyme deficient in Fabry disease in 10 of the 11patients. Eight patients in the study received an ascending dose of 25,100, and then 250 mg b.i.d. over 6 weeks, followed by 50 mg/day for theremainder of the study (represented by closed circles) (FIG. 7) Threepatients in the study received 150 mg of DGJ every other day throughoutthe entire study (represented by closed circles). For purposes ofcalculating the percentage of normal in the table, the level of α-GALthat is normal was derived by using the average of the levels of α-GALin white blood cells of 15 healthy volunteers from the multiple-dosePhase I study. The 11 patients represented 10 different geneticmutations and had baseline levels of α-GAL that ranged from zero to 30%of normal.

GL-3 Levels.

Kidney GL-3 levels were assessed by an independent expert using electronmicroscopy. Data available for two patients to date showed an observeddecrease in GL-3 in multiple cell types of the kidney of one patientafter 12 weeks of treatment (mesangial cells and cells of the glomerularendothelium and distal tubules). A second patient showed a decrease ofGL-3 levels in the same kidney cell types after 24 weeks of treatment,but these decreases were not independently conclusive because of thepatient's lower levels of GL-3 at baseline. Both patients showed adecrease of GL-3 levels in other kidney cell types including cells ofthe interstitial capillaries, but the decreases were less than 1 unitand, thus, even though the post-treatment. These initial results areconsistent with the GL-3 reductions observed after oral administrationof Amigal to mice that produce defective human α-GAL.

Skin GL-3 levels at baseline and after treatment as assessed by lightand electron microscopy are available for 10 patients. Seven patientshad skin GL-3 levels that were normal or near normal both before andafter treatment. Results for the three other patients were difficult tointerpret because they showed evidence of a decrease in GL-3 in someskin cell types and an increase in GL-3 in other skin cell types, withvariability over time.

Example 6: Dosing Regimens for the Treatment of Fabry Disease Using DGJHydrochloride

This example describes a study of DGJ (1-Deoxygalactonorjirimycin) inFabry patients.

Patient Enrollment.

Eighteen male and nine female Fabry patients with known missensemutations in α-GAL (verified by genotype) were enrolled. (One of themale patients did not complete the study). Thirteen of these patientswere naive to ERT, while fourteen patients previously received ERT(Fabrazyme®), but had discontinued ERT for 21-274 days prior to thestudy. The Fabry disease of the patients enrolled in this study wascaused by one of the following missense mutations in the Fabry gene:A143T, T411, A97V, M51K, G328A, S276G, L300P, L415P, P259R, R301Q,N215S, P205T, F295C, C94S, or R112C.

Study Design.

Nine male patients in the study (one male did not complete the study)received an ascending dose of 25, 100, and then 250 mg b.i.d. for 6weeks (2 weeks at each dosage level), followed by six weeks of 25 mgb.i.d. or 50 mg/day for the remainder of the study (Group A). Four malepatients received a single 150 mg Q.O.D for 12 weeks (Group B); whilefive male patients received 150 mg Q.O.D. for 24 weeks (Group C).Finally nine female patients were randomized to receive one of threedosages: 50, 150 or 250 mg Q.O.D for 12 weeks (Group D) (FIG. 8).

α-GAL Activity.

Enzymatic activity of α-Gal in leukocytes (white blood cells; WBCs) ofthe patients was measured as a percentage of the average α-Gal activityin white blood cells of 15 healthy volunteers. α-GAL activity wasassessed in fractionated tissue obtained from biopsies, and in bloodleukocytes and plasma (from blood collected at baseline and everymonth).

GL-3 Deposits.

Kidney biopsies were performed at baseline, 12 weeks and 24 weekspost-treatment and evaluated for GL-3 deposits in various renal cells.GL-3 presence in the tissue was examined histologically as well asthrough the use of mass spectroscopy. Light microscopic analysis ofkidney biopsies was performed, wherein the accumulation of GL-3 in thetissue was classified in a manner similar to the classification analysisperformed in Kidney International, Vol. 62 (2002), pp. 1933-1946 whichis hereby incorporated by reference. Cells were classified as containingno GL-3 accumulation (“0”); mild GL-3 accumulation (“1”); moderate GL-3accumulation (“2”); or severe GL-3 accumulation (“3”).

Urinalysis.

Urine was analysed at baseline and periodically every 2-6 weeks forGL-3.

Cardiac Anaylsis.

In addition to the biopsies described above, MRIs, electrocardiogramsand echocardiograms with strain rate evaluations was performed atbaseline and periodically throughout the study to assess cardiacmorphology (e.g., left ventricular hyertrophy) and cardiac function(e.g., ejection fraction and conduction/rhythm abnormalities).

Renal Analysis.

Renal function was evaluated using glomerular filtration rate (GFR).

Neuropathic Pain.

Patients self-reported changes in symptoms at the end of 12 or 24 weektreatment period to evaluate, inter alia, reduction in acroparaesthesia.This may be evidence of clearance of GL-3 in the microvasculature ofperipheral nerve cells.

Results

Male Patients

α-GAL Activity.

The α-Gal activity data from the eight male patients receiving treatmentaccording to the Group A protocol is shown in FIG. 9. Patients wereclassified as “good” responders if, following treatment, they exhibitedan absolute increase in enzyme activity that was greater than 3% ofnormal α-GA1 activity and further, such increase was greater than 33%relative to the mutant's pre-treatment α-GAL activity level. Patientswere classified as “moderate” responders if they exhibited an absoluteincrease greater than 1-3% of normal α-GAL activity that was alsogreater than 33% relative to the mutant's pre-treatment α-GAL activitylevel.

The data from the nine male patients receiving treatment according toprotocols Group B and Group C are shown in FIG. 10. “Good” responderswhere characterized by an increase in α-Gal activity to about 8% ofnormal enzyme activity by week 12 of the treatment. “Moderate”responders were those patients that exhibited an increase in α-Galactivity to about 1.5% normal enzyme activity by 24 weekspost-treatment. “Non” responders were those patients that neverexhibited an increase in α-Gal activity above 1% normal enzyme activityduring treatment.

There were eleven “good” responders in the study, while four patientswere “moderate” responders and two patients were “non” responders. Ofthe good responders, six patients had a residual α-Gal activity ofgreater than 3% of normal enzyme activity prior to initiation of thestudy, while five of the good responders and all of the moderate andnon-responders exhibited a residual α-Gal activity of less than 3%normal level (FIG. 11).

As shown in FIGS. 12 and 13, the eleven “good” responders exhibited amean 630% increase in WBC α-Gal activity when pre and post treatmentactivity levels of each patient were compared. Six of the goodresponders also showed a mean 1090% increase in kidney α-Gal activity.All four “moderate” responders displayed a mean 170% increase inleukocyte α-Gal activity during treatment, and one moderate responderexhibited a mean 100% increase in kidney α-Gal activity. None of the“non” responders exhibited any overall increase in either leukocyte orkidney α-Gal activity following treatment.

Urinalysis of GL-3.

GL-3 in the urine of treated patients results primarily from tubulecells shed from the kidneys. Elevated levels of GL-3 are detectable inall Fabry patients. In the male patients who were characterized as“good” responders, patients displayed a 38% mean decrease in urine GL-3levels following treatment, while eight of the eleven good respondersexperienced a decrease that was greater than 10%. While both the“moderate” responders and “non” responders showed overall increases inurine GL-3 following treatment, one patient in the moderate groupdisplayed a decrease in GL-3 levels that was greater than 10% followingtreatment (FIG. 14).

Kidney Analysis.

Kidney GL-3 levels were assessed using histological and massspectroscopic analysis. Kidney biopsies were examined in four of the“good” responders, two of the “moderate” responders, and two of the“non” responders. Accumulation of GL-3 was examined in three differentkidney cell types: interstitial capillaries, distal tubules, andpodocytes. With respect to the good responders, one patient displayed adecrease in interstitial capillary GL-3, one patient experienced anundetectable change in interstitial capillary GL-3, and one patientexperienced no change in interstitial capillariy GL-3. With regard todistal tubules, three of the good responders experienced a decrease inGL-3, and one good responder experienced an increase in GL-3. As forpodocyte cell GL-3 levels following treatment, all four good respondersexperienced no change GL-3 levels (See FIG. 15A).

With regard to overall GL-3 levels in the kidney biopsies, two of thegood responders showed a decrease in GL-3 following treatment, while twogood responders showed no change in GL-3 levels. As for the moderateresponders, two patients showed a decrease in GL-3 levels. Onenon-responders showed a decrease in GL-3 levels, while onenon-responding patient exhibited an increase in GL-3 (See FIG. 15B).

Additionally, as was seen in the urinalysis, mass spectroanalysis ofkidney biopsies revealed that the good responders experienced a meandecrease in kidney GL-3 levels (28%) following treatment, with 3 of thegood responders exhibiting a decrease of greater than 10% (FIG. 16).

Renal function was measured using glomerular filtration rate (GFR) (MDRDequation was used to estimate GFR using serum creatinine adjusted forage, gender and race). Approximately half of all Fabry patients have anabnormally low GFR (<90 ml/min/1.73 m²). Natural history studies suggestthat Fabry patients exhibit a progressive decline in GFR at a rate ofabout 5-15 units per year depending on age and kidney disease stage. Asshown in FIG. 17A-B, the good responders maintained a mean eGFR withinthe normal eGFR range of 90-120 ml/min/1.73 m² during the entiretreatment procedure (FIG. 17A), while the predicted mean eGFR level ofuntreated individual is projected to continue declining below 90ml/min/1.73 m² (FIG. 17B).

Cardiac Function.

Prior to treatment, about half of all the patients had conduction/rhythmabnormalities as assessed via ECG at baseline prior to treatment, and atthe last visit following treatment (the last visit was between 12-24weeks after the study began)(data not shown). Three of the “good”responders had an abnormally high left ventricle mass prior totreatment. One of these patients displayed an 8% decrease in leftventricle mass following 12 weeks of treatment, while 2 exhibited nochange in left ventricle mass after 48 weeks of treatment (data notshown). Both decreases in and maintenance of left ventricle mass is ofinterest since Fabry patients typically exhibit an increase in leftventricle mass over time. Furthermore, three of the good responderspresented an abnormal ejection fraction prior to treatment, wherein twoof the patients displayed an ejection fraction in the normal range(>55%) following treatment (FIG. 18).

Self-Reported Analysis.

Patients self-reported changes in symptoms, such as acroparaesthesiasassociated with Fabry disease, at end of the 12 or 24 week treatmentperiod, and every 12 weeks in extension. Seven of the “good” respondersreported improved gastrointestinal function and a decrease in pain;increases in the ability to walk, drive and sleep; and improvedsweating. Two of the good responders reported no change in Fabrysymptoms. Of the “moderate” and “non” responders, one person reportedincreased sweating with a persistence of pain, and three reported nochanges in Fabry symptoms (FIG. 19).

Female Patients

Because of X-chromosome inactivation in female cells, the phenotype of adiseased cell in a tissue sample expressing a mutant Fabry gene will bemasked by healthy cells in the sample. Therefore, to assess the expectedenzyme responses in diseased cells, each mutation the female patientsexhibited was constructed and tested in vitro. Thus, based on the invitro analysis, the different mutations were classified as “expectedgood responders” and “expected non-responders.” Five of the patientswere classified as expected good responders while four patients wereclassified as expected non-responders (data not shown).

α-GAL Activity.

All nine of the female patients treated in the study exhibited anincrease in WBC α-Gal activity following treatment according to theGroup D treatment protocol (mean increase of 146% compared to baselineenzyme levels prior to treatment) (data not shown).

Urinalysis of GL-3.

In the female patients who were characterized as “expected good”responders, the patients displayed a 20% mean decrease in urine GL-3levels following treatment, while 3 of the 5 expected good respondersexperienced a decrease that was greater than 10%. The “non” respondersshowed an overall increases in urine GL-3 levels following treatment,although one patient displayed a decrease in GL-3 that was greater than10% following treatment (FIG. 20).

Kidney Analysis.

Similar to the results observed in the urinalysis, mass spectoanalysisof kidney biopsies from the “expected good” responders displayed a meandecrease in kidney GL-3 levels (20%) following treatment, wherein two ofthe five expected good responders presented a decrease of greater than10% following treatment (FIG. 21).

Self-Reported Analysis.

Patients self-reported changes in symptoms, such as acroparaesthesiasassociated with Fabry disease, at end of the 12 or 24 week treatmentperiod, and every 12 weeks in extension. Four of the “expected good”responders reported decreases in pain; increases in the ability to walk,drive and sleep; and improved sweating. One of the expected goodresponders reported no change in Fabry symptoms. Of the “non”responders, one person reported a decrease in pain, while three reportedno changes in Fabry symptoms or the appearance of symptoms such asanxiety, depression, or sleep difficulties (FIG. 22).

Example 7: Treatment of Pompe Disease Using 1-Deoxynorjirimycin

100 mg/kg of 1-Deoxynorjirimycin is administered ad libitum to mice for28 days. α-glucosidase activity (GAA) in the heart is shown in FIG. 23for whole tissue lysates (left) and based on immunoprecipitated GAA(right). This data appears similar preliminary results fromgastrocnemius muscle analysis in response to 1-Deoxynorjirimycin.

1-Deoxynorjirimycin has been shown to be well tolerated in short-termsafety studies in rats and monkeys at doses currently believed to bewell above levels to be encountered in future clinical studies. Forexample, 1-Deoxynorjirimycin appears to be safe and well tolerated atsingle doses up to 600 mg. Repeat doses up to 2 grams per day for 2weeks. All adverse events in patients receiving drug were mild ormoderate in severity, and none were considered definitely or probablyrelated to the study drug. 1-Deoxynorjirimycin is believed to have highoral bioavailability with a terminal half-life in plasma ofapproximately 4-8 hours.

GAA response to 1-Deoxynorjirimycin will be determined in freshlyisolated leukocytes. GAA response will also be determined inpatient-derived cell lines, skin fibrolasts and EBV-transformedlymphoblasts. DNA sequencing will be performed to confirm genotypeinformation. Urinary tetra-saccharide levels in patients will also beassessed. Plasma cytokines and chemokines will be measured to identifypotential markers of disease to monitor in clinical trials.

Example 8: DGJ (1-Deoygalactonorjirimycin Hydrochloride) Increases theActivity of α-Galactosidase A (α-GAL)

This example describes a study of DGJ (1-Deoxygalactonorjirimycin)transgenic mice expressing a missense Fabry mutation. The example alsodescribes the study of DGJ's affect on cell lines expressing variousFabry missense mutations.

Transgenic mice expressing the R301Q Fabry missense mutation wereadministered DGJ ad libitum at 100 mg/kg for four weeks. Following theDGJ treatment, biopsies were taken of the skin, liver and kidney of thetreated animals. α-GAL expression was measured in the tissue biopsies,as was the concentration of GL-3. As shown in FIG. 24, α-GAL expressionwas increased in the skin, heart and kidney following treatment withDGJ. Additionally, the concentration of GL-3 in the sampled tissues wasreduced following DGJ treatment. Furthermore, as shown in FIG. 25,histological examination of renal tube sections and cardiac sectionshowed that the presence of GL-3 aggregates was reduced followingtreatment with DGJ.

Cell lines were constructed to express one of 75 Fabry missensemutations. DGJ was administered to each cell line, and α-GAL activitywas measured to determine if DGJ increased the activity of the mutantenzyme. As shown in FIG. 26, DGJ enhanced α-GAL activity in 47 of the 75cell lines (63%). Furthermore, of the 57 cell lines expressing a Fabrymissense mutation associated with “classic” Fabry disease, 34 of thecell lines (60%) exhibited an increase in enzymatic activity followingtreatment. 20 of the 75 cell lines expressed a missense mutationcorresponding to later-onset Fabry disease. Of these 20 cell lines, 19(95%) displayed an increase in α-GAL activity following treatment.

Example 9: GCase Response with Isofagomine Tartrate in Bone and BoneMarrow in Normal Mice

A single dose of 100 mg/kg of isofagomine tartrate was administered tonormal mice. GCase activity (F460/μg protein) was measured in the femurbone and in bone marrow for both the study group and an untreatedcontrol group. Results are shown in FIG. 27.

Example 10: Pharmacokinetic/Tissue Distribution of Isofagomine in Rats

A single dose of 600 mg/kg of isofagomine was administered to rats viaPO gavage. The concentration of isofagomine (μM) in plasma, liver,spleen and brain tissue was ascertained at regular time intervals atdosing (t=0) through 48 hours after dosing. The results are shown inFIG. 28.

All tissues attained isofagomine levels exceeding the GCase enhancementEC₅₀ of about 400 μM within 15 minutes. Isofagomine levels fall belowthe GCASE Ki value after 48 hours in liver and plasma; spleen and braintissue showed a slower clearance.

Example 11: Comparison of DGJ Dosing Regimens in Male HR301Q GLA Tg/KOMice

Eight-week old male hR301Q GLA Tg/KO mice were treated for 4 weeks with300 mg/kg of DGJ in drinking water either daily (without washout period)or “less frequent” (4 days on/3 days off). Lysates were prepared fromskin, heart, kidney and plasma. GL-3 levels were measured by LC-MS/MS(expressed in mg/g tissue weight or mg/mL plasma). The results are shownin FIG. 29. LC-MS/MS data showed a greater reduction in GL-3 levels (*p<0.05 vs. untreated; #p<0.05 daily vs. less frequent, t-test) with lessfrequent DGJ dosing in tissues as well as plasma. Each bar representsthe mean±SEM of 10-16 mice/group.

Immunohistochemical staining with a monoclonal anti-GL-3 antibody(nuclei counterstained with methyl green) was also performed. Resultsare shown in FIG. 30 and shows GL-3 signal as dark red/brown spots(black arrows) in skin (fibroblasts and smooth muscle cells of bloodvessel wall), heart (smooth muscle cells of blood vessel wall), andkidney (distal tubular epithelial cells). Both daily and “less frequent”DGJ treatment reduced the amount and intensity of GL-3 signal in eachtissue (20×). Similar to LC-MS/MS, a greater GL-3 reduction was seen ineach tissue with less frequent DGJ dosing. Data shown are representativepictures from 7-8 mice/group.

Example 12: Comparison of DGJ Dosing Amounts in Male HR301Q GLA Tg/KOMice

Eight-week old male hR301Q GLA Tg/KO mice were treated for 4 weeks with3, 10, 30, 100, or 300 mg/kg/day of DGJ in drinking water. Tissuelysates from skin, heart, and kidney were prepared and tested for GLAactivity (using 4-MUG as substrate, expressed in nmol/mg protein/hr),GLA protein (using immunoblotting of 50 mg tissue lysate with anti-humanGLA antibody) and GL-3 levels (using LC-MS/MS, expressed in mg/g oftissue weight). Results are shown in FIG. 31. A significant anddose-dependent increase in GLA activity (* p<0.05 vs. untreated, ANOVA)and GLA protein (inset, GLA runs as ˜45 kD band) and a significantreduction in GL-3 levels (* p<0.05 vs. untreated, ANOVA) were seen afterDGJ treatment. Each bar represents the mean±SEM of n=7-8 mice/group.Each lane in the Western blots represents one mouse from each group.

Example 13: Half-Life Determination of DGJ and Elevated HR301Q GLA INMale HR301Q GLA Tg/KO Mice

Half-lives of elevated hR301Q GLA and DGJ were estimated by dosinghR301Q GLA Tg/KO male mice for 4 weeks with 100 mg/kg/day of DGJ(drinking water), followed by 7 day washout (without DGJ in drinkingwater). Mice were euthanized at 0, 1, 3, 5, and 7 days after DGJwithdrawal and GLA levels (solid line in skin, heart and kidney) weremeasured using 4-MUG. Concentrations of DGJ were measured by LC-MS/MS(dotted line in skin, heart, and kidney) simultaneously. The results areshown in FIG. 32.

Using exponential decay curves, the half-life of elevated tissue hR301QGLA levels was estimated as 2-2.5 days, while that of DGJ was estimatedat 6-7 hours. Each data point represents the mean±SEM of 6-7 mice/group.

Example 14: Half-Life Determination of DGJ and Elevated HR301Q GLA INMale HR301Q GLA Tg/KO Mice

Oral administration of DGJ to healthy male volunteers (50 and 150 mgtwice daily for 7 days; n=6 for treatment groups, n=4 for all placebo)resulted in increased GLA levels, as measured by 4-MUG in white bloodcell lysates. DGJ was orally available and was generally well-toleratedat all doses, with no serious adverse events occurring in any treatmentgroup. Data were normalized to the predose values of each group (predosevalues are 24.6, 23.3, and 14.1 nmoles/mg protein/hr for placebo, 50 and150 mg respectively). Results are shown in FIG. 33.

Example 15: Dosing Regimens for the Treatment of Fabry Disease Using DGJHydrochloride

This example describes a study of DGJ (1-deoxygalactonorjirimycin) inFabry patients.

Patient Enrollment.

Eligible patients were 16-74 years old and had genetically-confirmedFabry disease, had either never received or had not received enzymereplacement therapy for >6-months, had a GLA mutation that resulted in amutant protein that would respond to DGJ, based on the human embryonickidney-293 (HEK) assay used at the time of enrollment, had an eGFR >30ml/minute/1.73 m², and had a urinary GL-3>4 times the upper limit ofnormal.

Study Design.

Following eligibility-baseline assessments (2-months), patients wererandomized to Stage 1—6 months of double-blind administration of 150 mgDGJ HCl or placebo every other day. All patients completing Stage 1 wereeligible to receive open-label DGJ in Stage 2 (months 6-12) and for anadditional year (months 13-24) thereafter (AT1001-011/NCT00925301). Theprimary objective was to compare the effect of DGJ to placebo on kidneyGL-3 as assessed by histological scoring of the number of inclusions ininterstitial capillaries after 6 months of treatment. The secondaryobjectives of Stage 1 were to compare the effect of DGJ to placebo onurine GL-3 levels, on renal function, 24-hours urinary protein, and onsafety and tolerability. The tertiary objectives were cardiac function,patient-reported outcomes, exploratory kidney analyses, and white bloodcell α-galactosidase activity. Study completers were eligible to enrollin the open-label study—AT1001-041/NCT01458119—for up to 5 years.

Kidney Histology Assessment.

Each patient underwent a baseline kidney biopsy, as well as repeatkidney biopsies at 6 and 12 months. The number of GL-3 inclusions perkidney interstitial capillary per patient at baseline, and at 6 and 12months was quantitatively assessed in 300 capillaries by 3 independentpathologists blinded to treatment and visit. All values for eachindividual biopsy at a given time were averaged prior to statisticalanalysis.

GL-3 changes in podocytes, endothelial cells, and mesangial cells, andglomerular sclerosis, were assessed qualitatively by the same 3pathologists blinded to treatment/visit.

Globotriaosylceramide and Globotriaosylsphingosine.

Plasma lyso-Gb3 and 24-hour urine GL-3 were analyzed by liquidchromatography-mass-spectroscopy using a novel stable isotope-labeledinternal standard, 13C6-lyso-Gb3 (lower-limit-of-quantification: 0.200ng/mL, 0.254 nmol/L).

Renal Function Assessment.

Annualized rates of change (mL/min/1.73 m²/year) were calculated usingChronic Kidney Disease Epidemiology Collaboration-eGFRCKD-EPI) andmeasured iohexol clearance-mGFRiohexol).

Echocardiography.

Left ventricular mass index, left posterior wall thickness, diastolic,interventricular septum thickness, diastolic and other parameters wereassessed through blinded, centralized evaluation. The baseline visit ofextension study AT1001-041/NCT01458119 was used as the last assessment.

Patient-Reported Outcomes.

Patient-reported outcomes were assessed using theGastrointestinal-Symptoms-Rating-Scale (GSRS), Short Form-36v2TM andBrief-Pain-Inventory-Pain-Severity-Component.

Safety Analysis and Adverse Events.

Randomized patients receiving ≥1 dose were included in the safetyanalysis, which comprised vital signs, physical exams,electrocardiograms, clinical labs, and adverse events.

Statistical Analyses for Kidney Interstitial Capillary GL-3 Substrate.

The primary Stage 1 (6 month) endpoint (ITT population with baselinebiopsies, n=64) was the proportion of patients in the DGJ and placebogroups with a ≥50% reduction in GL-3 inclusions per interstitialcapillary. Two other Stage 1 endpoints were assessed (modified-ITTpopulation: randomized patients with paired baseline and month 6biopsies; n=60): percent change in GL-3 inclusions per interstitialcapillary, and percent of interstitial capillaries with zero GL-3inclusions.

Efficacy analyses for GL-3 inclusions per interstitial capillary andother pre-specified endpoints in Stage 2 (months 6-12) and theopen-label-extension (months 12-24) were based on the modified intentionto treat (mITT)—population consisting of randomized patients with mutantα-galactosidase enzyme shown to be suitable for DGJ treatment by thevalidated assay; n=50).

Results

Baseline Characteristics.

Sixty-seven patients (16-74 years-old; 64% female) with potentiallyresponsive mutant α-galactosidase were randomized (ITT population).Table 6 provides the baseline characteristics for the 50 patients in theITT population with suitable mutant α-galactosidase. There were nostatistically significant differences in baseline parameters.

TABLE 6 Treatment Group Placebo to DGJ DGJ HCl HCl Total Parameter (N =28) (N = 22) (N = 50) Age (year) (n) 28 22 50 Mean ± SD  41.5 ± 13  45.1± 8.0  43.1 ± 11 Median 37.0 45.5 45.0 Weight (kg) (n) 28 22 50 Mean ±SD  72.6 ± 15.35  76.1 ± 16.52  74.1 ± 15.81 Median 72.3 74.0 72.8Number of Years of Diagnosis of 28 21 49 Fabry Disease (n) Mean ± SD 5.6 ± 6.89  7.3 ± 8.80  6.3 ± 7.73 Median  4.1  4.1  4.1 Number ofpatients previously on ERT  4 (14.3%)  7 (31.8%) 11 (22.0%) (>6-monthsprior to baseline) (%) Use of ACEi/ARB/Ri at Baseline Yes (%)  9 (32.1%)12 (54.5%) 21 (42.0%) No (%) 19 (67.9%) 10 (45.5%) 29 (58.0%)Proteinuria >150 mg/24 h (%) 17 (60.7%) 18 (81.8%) 35 (70.0%)Proteinuria >300 mg/24 h (%)  8 (28.6%) 11 (50.0%) 19 (38.0%)Proteinuria >1000 mg/24 h (%)  3 (10.7%)  3 (13.6%)  6 (12.0%)mGFR_(Iohexol) (mL/min/1.73 m²) (n) 27 21 48 Mean ± SD 79.95 ± 30.983.12 ± 22.8 81.34 ± 27.5 Median 84.90 82.20 83.40 eGFR_(CKD-EPI)(mL/min/1.73 m²) 28 22 50 Mean ± SD  94.4 ± 27.0  90.6 ± 17.1  92.7 ±23.0 Median 96.6 93.5 94.0 Lyso-Gb₃ (n) 18 13 31 Mean (nmol/L) ± SD 47.3 ± 62  41.9 ± 39  45.0 ± 53

Published reports of clinical phenotype(s) associated with the genotypesof patients with suitable mutations (n=50) indicate that 30 (60%) hadmutations associated with the classic phenotype of Fabry disease, one(2%) with the non-classic phenotype, three (6%) with both phenotypes,and 16 (32%) not yet classified. Residual WBC α-galactosidase activity≤3% was found in 14 of 16 (87%) males; 29 of 31 (94%) males and femaleshad elevated plasma lyso-Gb3, and 47 of 50 (94%) males and females hadmulti-organ system disease.

DGJ and Kidney Interstitial Capillary GL-3.

In the 6-month primary outcome analysis (ITT), 13 of 32 (41%) DGJ and 9of 32 (28%) placebo-treated patients achieved a response (>50% reductionin GL-3 inclusions per interstitial capillary) (p=0.30). The medianchange in interstitial capillary GL-3 from baseline was −40.8% for DGJversus −5.59% for placebo (p=0.097). The mean difference for the changein % of interstitial capillaries with zero GL-3 inclusions was 7.3% infavor of DGJ (p=0.042).

In Stage 1 (6-month post hoc) and Stage 2 (12-month prespecified)analyses (mITT—suitable population; n=45), 6 months of DGJ wasassociated with a significantly greater reduction in interstitialcapillary GL-3 (±SEM) compared to placebo: −0.250±0.103 versus+0.071±0.126; p=0.008. The reduction in interstitial capillary GL-3 at 6months remained stable following an additional 6 months of treatment. Asignificant reduction in interstitial capillary GL-3 (±SEM) was observedat 12-months in patients switching from placebo to DGJ at 6 months(−0.330±0.152; p=0.014). Patients with mutant α-galactosidase that wasnot suitable for DGJ therapy according to the validated assay did notshow any treatment effect in interstitial capillary GL-3.

DGJ and GL-3 in Glomerular Cells.

Based on qualitative assessments on 23 kidney biopsies, following 12months of DGJ, patients with responsive mutant α-galactosidase showeddecreases in glomerular podocyte (5 of 23 biopsies; 22%), endothelialcell (6 of 23 biopsies; 26%), and mesangial cell GL-3 (11 of 23biopsies; 48%). None of the samples had increases; the remaining samplesshowed no change.

DGJ and Plasma Lyso-Gb3 Levels.

Six months of DGJ (mITT-suitable) was associated with a significantreduction in plasma lyso-Gb3 levels compared to placebo (p=0.0033).Plasma lyso-Gb3 remained stable without further reduction following 6additional months of DGJ. A significant reduction in plasma lyso-Gb3 wasfound in patients (ITT-suitable) switching from placebo to DGJ between 6and 12-months (p<0.0001). Plasma levels in patients with mutantα-galactosidase that was not suitable were unchanged.

DGJ and Urine GL-3 Substrate.

In patients with suitable mutant α-galactosidase, mean changes in24-hour urine GL-3 substrate (±SEM) concentration for DGJ and placebo(baseline to month 6) were: −361±169 (to 555±151) and −147±217 (to1017±218) ng/mg creatinine, respectively (p=0.44).

DGJ and Kidney Function. There were no statistically significantdifferences between the DGJ and placebo arms in eGFRCKD-EPI, ormGFRIohexol changes from baseline to month 6 (mITT-suitable).

In patients followed for up to 24 months of DGJ (mITT-suitable), theannualized changes in eGFRCKD-EPI and mGFRiohexol (±SEM) were −0.30±6.6,and −1.51±1.33 mL/min/1.73 m², respectively. Male gender and higherbaseline proteinuria were associated with higher rate of annual decline.There were no statistically significant differences in baseline levelsor changes from baseline between treatment groups for 24-hour urineprotein.

DGJ and Echocardiographic Parameters.

At baseline, left-ventricular-mass-index was comparable between groupswith no significant differences in Stage 1.

In patients (ITT-suitable), who received DGJ for up to 24 months, astatistically significant decrease in left-ventricular-mass-index (LVMi)(p<0.05 based on the 95% CI not including 0) was observed overall with atrend toward a larger reduction in patients with baseline LVhypertrophy. Table 7 shows the echocardiographic-derived LVMi changesfrom baseline to month 18/24 for ITT-suitable patients.

TABLE 7 Patients with Change from Baseline to Suitable Mutant Baseline²Month 18/24³ α-galactosidase¹ Mean ± SEM (g/m²) Mean ± SEM (95% CI) All 96.5 ± 5.0 −7.69 ± 3.7 (−15.4, −0.009)⁴ n = 44 n = 27 Patients with LVH138.9 ± 11 −18.6 ± 8.3 (−38.2, 1.04) at baseline n = 11 n = 8 LVMi,Left-ventricular-mass-index (g/m²): Normal: 43-95 (female), 49-115(male); LVH, left ventricular hypertrophy; ¹Includes patients with abaseline and post-baseline ECHO, who received ≥18-months DGJ. ²Month 6used as baseline for placebo patients switching to DGJ; Baseline used ifno month 6. ³Baseline of extension study used as month 18/24.⁴Statistically significantly different from baseline based on 95% CIsnot overlapping with 0; p < 0.05

Interventricular septal wall thickness decreased by 0.061 cm±0.051(5.2%) from baseline (1.17 cm±0.057) (95% CI: −1.67, 0.045); the leftventricular posterior wall thickness was stable for up to 24 months. Thechanges in left-ventricular-mass-index correlated with changes in IVSWT(R2=0.26, p=0.006) but not with changes in left ventricular posteriorwall thickness (R2=0.06, p=0.230).

LVMi continued to decrease over 30/36 months of treatment in anextension of this study [change from baseline (±SD): −7.8 g/m²±21.5]. Inpatients with baseline LVH (n=4), the change from baseline was larger,−30.0±17.5 g/m². The LVMi changes from baseline to after 6/12, 18/24 and30/36 months of DGJ therapy are shown in FIG. 34.

Gastrointestinal Symptoms Rating Scale.

Gastrointestinal symptoms improved in 3 of 5 domains (diarrhea, reflux,indigestion) in DGJ-treated ITT-suitable patients, as shown in Table 8below.

For the diarrhea domain, between baseline and month 6 (Stage 1), therewas a statistically significant decrease (p=0.03; ITT-suitable); anonsignificant decrease was also observed for ITT-suitable patients withbaseline symptoms (p=0.06). Statistically significant changes over 24months were found for ITT-suitable patients and ITT-suitable patientswith baseline symptoms (p<0.05, based on the 95% CI not including 0).

There was a statistically significant improvement in the reflux domainin Stage 1 in ITT-suitable patients with baseline symptoms (p=0.047).Statistically significant changes over 24 months were found in theindigestion domain for ITT-suitable patients and ITT-suitable patientswith baseline symptoms (p<0.05 based on the 95% CI not including 0).There was a trend toward improvement in the constipation domain.

TABLE 8 Changes in Gastrointestinal Symptoms Rating Scale¹(ITT-Suitable) GSRS Domain Abdominal Treatment Diarrhea RefluxIndigestion Constipation Pain Group DGJ Placebo DGJ Placebo DGJ PlaceboDGJ Placebo DGJ Placebo Mean Baseline Values (n) All patients 2.3 2.11.4 1.4 2.5 2.4 1.9 2.0 2.1 2.3 (28)   (22)   (28)   (22)   (28)  (22)   (28)   (22)   (28)   (22)   Patients 3.2 3.1 2.1 2.6 2.8 2.7 2.52.4 2.4 2.9 with (17)   (11)   (10)   (6)   (23)   (19)   (17)   (15)  (22)   (15)   Symptoms at BL Change from Baseline to Month 6 (Stage 1,Double-Blind) All Patients −0.3*² +0.2  −0.1   +0.2  −0.1   −0.1   +0.1 +0.2  0.0 0.0 Patients −0.6   +0.2  −0.6*³ +0.6  −0.2   −0.2   +0.2 +0.1  −0.1   −0.1   with Symptoms at BL Change from Baseline (DGJ) orMonth 6 (Placebo) to Month 24 (OLE DGJ Treatment) All Patients −0.5(−0.9, −0.1)*⁴ −0.2 (−0.5, 0.2) −0.4 (−0.7, −0.04)*⁴ −0.4 (−0.7, +0.0)*⁵−0.2 (−0.5, +0.1) Patients −1.0 (−1.5, −0.4)*⁴ −0.6 (−1.5, 0.2) −0.5(−0.8, −0.06)*⁴ −0.5 (−1.1, +0.0)*⁵ −0.2 (−0.6, 0.1)  with Symptoms atBL *Indicates significant or borderline significant changes frombaseline. ¹Least squares means for change from baseline (BL)|²p = 0.03and ³p = 0.047 using ANCOVA|⁴Statistically significant or ⁵Trend basedon 95% CIs with the upper bound of 0.

DGJ and Podocyte GL-3. Kidney biopsy samples from enzyme replacementtherapy-naïve male patients with Fabry disease with GLA mutationsamenable to DGJ (N=8), taken at baseline and again after 6 months of DGJtreatment, were studied by masked unbiased electron microscopystereology. The mean±SD V(Inc/PC) of all patients decreased from2568±1408 μm³ at baseline to 1282±792 μm³ after 6 months of DGJ(p=0.0182), as shown in FIG. 35. There was a correlated reduction inmean podocyte volume from 6680±2835 μm³ at baseline to 3525±2084 μm³(p=0.004) after 6 months of DGJ (r=0.98, p=0.00003), as shown in FIG.36. These findings indicate that the podocyte cytoplasmic shrinkage wasproportional to GL-3 loss; thus, the volume fraction of podocytecytoplasm attributable to GL-3 did not change significantly. Themagnitude of podocyte GL-3 volume reduction following DGJ correlatedwith improvement of foot process width (r=0.82, p=0.02), as shown inFIG. 37. Mean plasma lyso-Gb3 also decreased from 118±48 nM at baselineto 75±42 nM after 6 months of DGJ (p=0.0004), as shown in FIG. 38. Thisdecrease correlated with % reduction in podocyte GL-3 volume (r=0.79,p=0.02). There was a trend between decrease in podocyte GL-3 volume andproteinuria (r=0.69, p=0.06) following treatment with DGJ for 6 monthsas shown in FIG. 39, but no association was found with glomerularfiltration rate. In this study, DGJ treatment was associated with a lossof GL-3 inclusions in podocytes in patients with Fabry disease. Thesensitive quantitative method used can assess treatment efficacy forthis important cell type over a relatively short period of time. Thismethod is also more sensitive than the methods described above in thekidney analysis of Example 5 (as shown in FIG. 14), as well as themethods earlier in this example relating to qualitative assessment ofpodocyte GL-3.

Safety and Adverse Events.

During Stage 1, the treatment-emergent adverse events were similarbetween groups. Adverse events with a higher frequency in patientsreceiving DGJ compared to placebo were headache (12/34 patients—35%versus 7/33 patients—21%) and nasopharyngitis (6/34 patients—18% versus2/34—6%). The most frequently reported adverse events for Stage 2 wereheadache (9/63 patients—14%) and procedural pain (7/63patients—11%—related to kidney biopsies) and, for theopen-label-extension, proteinuria (9/57 patients—16%), headache (6/57patients—11%), and bronchitis (6/57 patients—11%). Most adverse eventswere mild or moderate in severity. No adverse events led to DGJdiscontinuation.

Six patients experienced serious adverse events during Stage 1 (2: DGJ;4: placebo), 5 during Stage 2, and 11 during the open-label-extension.Two serious adverse events were assessed as possibly related to DGJ bythe investigator—fatigue and paresthesia. Both occurred in the samepatient between months 12-24 and resolved. No individual serious adverseevent was reported by >1 patient. Two patients discontinued DGJ due toserious adverse events; both were deemed unrelated to DGJ. No deathswere reported.

Treatment-emergent proteinuria was reported in 9 patients (16%) betweenmonths 12-24, and in one case, was judged as DGJ-related. In 5 patients,the 24-month values were in the same range as baseline. Three patientswith suitable mutations had overt baseline proteinuria (>1 g/24-hr),which increased over 24-months. In 23/28 patients with baselineproteinuria <300 mg/24-h, 24-hour urine protein remained stable duringDGJ treatment.

There was no progression to end-stage renal disease, no cardiac deathand no stroke as defined in Banikazemi et al. There was a single case oftransient ischemic attack—judged unrelated to DGJ.

Analyses of vital sign, physical findings, laboratory, and ECGparameters did not reveal any clinically relevant effect of DGJ.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

What is claimed is:
 1. A method of treating Fabry disease, the methodcomprising administering a capsule comprising 100 to 250 mg of acompound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof, wherein the patient hasan estimated glomerular filtration rate (eGFR)<90 ml/min/1.73 m².
 2. Themethod of claim 1, wherein the compound comprises:


3. The method of claim 1, wherein the compound comprises:


4. The method of claim 1, wherein the compound comprises:


5. The method of claim 1, wherein the compound comprises:


6. The method of claim 1, wherein the compound comprises:


7. The method of claim 1, wherein the capsule further comprises one ormore pharmaceutically acceptable excipients selected from the groupconsisting of lactose, starch, talc, magnesium stearate and silica. 8.The method of claim 1, wherein the capsule further comprises one or morepharmaceutically acceptable excipients selected from the groupconsisting of lactose, talc, magnesium stearate and silica.
 9. Themethod of claim 1, wherein the capsule further comprises lactose andtalc.
 10. The method of claim 1, wherein the capsule further comprisesstarch and magnesium stearate.
 11. The method of claim 1, wherein thecapsule is a gelatin capsule filled with the compound andpharmaceutically acceptable excipients consisting of lactose and talc.12. The method of claim 1, wherein the capsule is a gelatin capsulefilled with the compound and pharmaceutically acceptable excipientsconsisting of magnesium stearate and starch.
 13. The method of claim 1,wherein the total daily dose is up to 500 mg.
 14. The method of claim 1,wherein the capsule is administered twice a day.
 15. The method of claim1, wherein the capsule is administered every other day.
 16. The methodof claim 1, wherein the total daily dose is up to 500 mg and the patienthas an estimated glomerular filtration rate (eGFR)<90 ml/min/1.73 m².17. A method of treating Fabry disease, the method comprisingadministering a capsule comprising 100 to 250 mg of a compound selectedfrom the group consisting of

or a pharmaceutically acceptable salt thereof, wherein the patient hasan α-galactosidase A mutation selected from the group consisting ofL32P, N34S, T41L, M51K, E59K, E66Q, I91T, A97V, R100K, R112C, R112H,F113L, G132R, A143T, G144V, S148N, D170V, C172Y, G183D, P205T, Y207S,Y207C, N215S, R227X, R227Q, S235C, D244N, P259R, N263S, G271C, S276G,M284T, W287C, I289F, F295C, M296V, L300P, V316E, N320Y, G325D, G328A,R342Q, E358A, E358K, R363C, R363H, and P409A.
 18. The method of claim17, wherein the patient has an α-galactosidase A mutation selected fromthe group consisting of L32P, N34S, T41L, M51K, E59K, I91T, A97V, R112H,F113L, A143T, G144V, G183D, P205T, Y207S, N215S, D244N, P259R, N263S,M284T, F295C, M296V, L300P, G328A, E358A, R363C, R363H, and P409A.
 19. Amethod of treating Fabry disease, the method comprising administering acapsule comprising 100 to 250 mg of a compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof, wherein the total dailydose is up to 500 mg and the patient has an estimated glomerularfiltration rate (eGFR)<90 ml/min/1.73 m², wherein the patient has anα-galactosidase A mutation selected from the group consisting of L32P,N34S, T41L, M51K, E59K, I91T, A97V, R112H, F113L, A143T, G144V, G183D,P205T, Y207S, N215S, D244N, P259R, N263S, M284T, F295C, M296V, L300P,G328A, E358A, R363C, R363H, and P409A.
 20. The method of claim 19,wherein the capsule is administered twice a day.